Muskmelon (Cucumis melo) hydroperoxide lyase and uses thereof

ABSTRACT

The present invention provides a fatty acid lyase, wherein the activity of the lyase for 9-hydroperoxide substrates is greater than the activity for 13-hydroperoxide substrates and wherein K m  and V max  of the lyase for 9-hydroperoxylinolenic acid are greater than K m  and V max  of the lyase for 9-hydroperoxylinoleic acid. More particularly, the invention provides a lyase present in melon ( Cucumis melo ). The invention also provides a nucleic acid encoding the lyase, vectors, and expression systems with which the recombinant lyase can be obtained. The invention also provides methods of using the lyase of the invention, including methods of cleaving 9-hydroperoxylinoleic acid, 9-hydroperoxylinolenic acid, 13-hydroperoxylinoleic acid, and 13-hydroperoxylinolenic acid. Also, the invention provides a method of preparing 3-(Z)-noncnal, (3Z,6Z)-nonadienal, 2-(E)-nonenal, (2E,6Z)-nonadienal, or their corresponding alcohols and a method of preparing n-hexanal, 3-(Z)-hexen-1-al, 2-(E)-hexen-1-al, or their corresponding alcohols using the lyase of the present invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a fatty acid hydropcroxide lyaseprotein, which has activity for 9-hydroperoxide substrates and which ispresent in muskmelon (Cucumis melo), and the gene encoding the protein.The present invention also relates to the means for expressing thehydroperoxide lyase and methods of using the lyase in the field oforganic synthesis.

2. Background Art

Plants produce various volatile compounds that give rise to thecharacteristic flavors and odors of the particular plant. Unsaturatedfatty acids like linoleic and linolenic acids are precursors of flavorcompounds such as n-hexanal, hexan-1-ol, 2(E)-hexen-1-al,2(E)-hexen-1-ol, 3(Z)- hexen-1-al, 3(Z)- hexen-1-ol (also known aspipol), 3-(Z)-nonenal, (3Z,6Z)-nonadienal, 3-(Z)-nonenol,(3Z,6Z)-nonadienol, 2-(E)-nonenal, (2E,6Z)-nonadienal, 2-(E)-nonenol,and (2E,6Z)-nonadienol. These compounds are used widely in flavors,particularly fruit flavors, and are used by the aroma industry for afruit aroma. The demand for these flavor compounds has grown to exceedtheir supply from traditional sources, thus motivating research effortstoward finding alternative natural ways of obtaining these materials.

The synthesis of these flavor compounds starts from free(polyunsaturated) fatty acids such as linoleic (9(Z),12(Z)-octadecadienoic) and α-linolenic (9(Z), 12(Z), 15(Z)-octadecatrienoic) acids. In nature, these acids are released from cellmembranes by lipolytic enzymes after cell damage. Fatty acidhydroperoxides are formed by the action of a lipoxygenase (LOX) and aresubsequently cleaved by a hydroperoxide lyase to give C₆- andC₉-volatile flavor compounds together with ω-oxoacids. The cleavage of13-hydroperoxides yields C₆-compounds, including hexanal and(3Z)-hexenal, and the cleavage of 9-hydroperoxides yields C₉-compounds,(3Z)-nonenal and (3Z,6Z)-nonadienal. In the presence of isomerases,these aldehydes are isomerized to (2E)-enals. Furthennore, alcoholdehydrogenases can convert the aldehydes into their correspondingalcohols.

The HPL enzymes have proven difficult to study because they are membranebound and are present in only small quantities in plant tissue. The HPLenzymes have been characterized as 13-HPLs or 9-HPLs, according to theirsubstrate specificity. The 13-HPL enzyme was identified for the firsttime in banana fruits (Tress1 and Drawert, 1973) and was subsequentlystudied in a number of different plant materials, including watermelonseedlings (Vick and Zimmerman, 1976), apple and tomato fruits (Schreierand Lorenz, 1982), tomato leaves (Fauconnier et al., 1997), cucumberseedlings (Matsui, et al, 1989), and soybean seedlings (Olias et al.,1990). The 13-HPL enzyme has been purified from tea leaves (Matsui etal., 1991) and, more recently, from green bell pepper fruits (Shibata etal., 1995), tomato leaves (Fauconnier et al., 1997), sunflower (Itoh andVick, 1999), guava (PCT application, WO 9958648 A2), and banana(European Patent Application, Publication No. EP 0801133 A2). A9-hydroperoxide specific HPL has been identified in pear (Kim andGrosch, 1981). There have been studies that suggested the presence of athird type of HPL that cleaves both 9- and 13-hydroperoxides. (Matsui etal. 1989; Hornostaj and Robinson, 1998).

Crude sources of lyases are currently used in an industrial process forthe production of flavors and aromas. (See, e.g., U.S. Pat. No.5,464,761). In this process, a solution of the required substrates madefrom linoleic or linolenic acid (obtained from sunflower and linseedoils, respectively) using freshly prepared soybean flour as a source ofLOX. This solution is then mixed with a freshly prepared puree of wholefruit, as the crude source of HPL. The aldehyde products are thenisolated by distillation. When the alcohols are required, fresh baker'syeast is added to the hydroperoxide solution before it is mixed with thefruit puree. This yeast contains an active alcohol dehydrogenase enzymethat reduces the aldehydes as they are formed by the HPL.

There are a number of disadvantages to this industrial process. Theprincipal disadvantage is the requirement of large quantities of freshfruit. Such a requirement means that the process has to be operated in acountry where fresh fruit is cheaply and freely available. Even whensuch a site is found, availability is limited to the growing season ofthe fruit.

A second disadvantage is that the desired enzyme activities are ratherdilute in the sources employed. This means that relatively large amountsof soy flour, fruit puree, and yeast have to be used in the process. Thelarge volumes of these crude materials that are required for industrialproduction place physical constraints on the yields of flavor and aromacompounds that can be achieved.

A third disadvantage is that it is a large-volume batch process, which,by its nature, does not make maximum use of the HPL's catalyticactivity, is relatively labor intensive, and generates a large amount ofresidual organic material. The residual organic material mustsubsequently be transported to a compost farm or otherwise discarded.

The present invention overcomes these limitations and disadvantagesrelated to the source of muskmelon 9-HPL by providing purified andrecombinant muskmelon 9-HPL proteins, nucleic acids, expression systems,and methods of use thereof.

SUMMARY OF THE INVENTION

The present invention provides a fatty acid lyase and a nucleic acidencoding the lyase. In particular, an isolated fatty acid hydroperoxidelyase is disclosed, wherein the activity of the lyase for9-hydroperoxide substrates is greater than the activity for13-hydroperoxide substrates and wherein K_(m) and V_(max) of the lyasefor 9-hydroperoxylinolenic acid are greater than K_(m) and V_(max) ofthe lyase for 9-hydroperoxylinoleic acid. More particularly, theinvention provides a lyase present in melon (Cucumis melo), and anucleic acid encoding the lyase. The invention also provides a vector,comprising the nucleic acid of the invention, and expression systemswith which the recombinant lyase can be obtained.

The invention also provides methods of using the lyase of the invention,including a method of cleaving a (9S, 10E, 12Z) 9-hydroperoxyoctadeca-10,12-dienoic acid or (9S, 10E, 12Z, 15Z) 9-hydroperoxyoctadeca-10,12,15-trienoic acid into a C9-aldehyde and a C9-oxononanoic acid anda method of cleaving (9Z, 11E, 13S) 13-hydroperoxyoctadeca -9,11-dienoicacid or (9Z, 11E, 13S, 15Z) 13-hydroperoxyoctadeca-9,11,15-trienoic acidinto a C6- aldehyde and a C12-oxocarboxylic acid. Also, the inventionprovides a method of preparing 3-(Z)-nonenal, (3Z,6Z)-nonadienal,2-(E)-nonenal, (2E,6Z)-nonadienal, or their corresponding alcohols from(9S, 10E, 12Z) 9-hydroperoxyoctadcca-10,12-dienoic acid or (9S, 10E,12Z, 15Z)9-hydroperoxyoctadeca- 10,12,15-trienoic acid using the lyaseof the present invention. Also provided is a method of preparingn-hexanal, 3-(Z)-hexen-1-al, 2-(E)-hexen-1-al, or their correspondingalcohols from (9Z, 11E, 13S) 13-hydroperoxyoctadeca-9,11-dienoic acid or(9Z, 11E, 13S, 15Z) 13-hydroperoxyoctadeca-9,11,15-trienoic acid usingthe lyase of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the full length amino acid sequences for Guava-HPL,Banana-HPL, Pepper-HPL, Arab-AOS, Flax-AOS, Guayule-AOS, Melon AOS, andthe Melon 9-HPL with the regions having a high degree of identity shownin dark boxes and the consensus sequence labeled as “majority.”

FIG. 2A is a schematic showing the melon cDNA and the regions where thedegenerate primers, based on other HPLs and AOSs, bound to produce boththe 150 bp and 70 bp cloned products from melon.

FIG. 2B shows an alignment of partial amino acid sequences fromGuava-HPL, Banana-HPL, Pepper-HPL, Arab-AOS, Flax-AOS, and Guayule-AOS.The boxed regions represent areas of high homology among HPLs and AOSs.

FIG. 3 shows the sequences of the degcnerate primers used to obtain the150 bp and 70 bp fragments of melon HPL and AOS.

FIG. 4 shows the amino acid sequence alignment of three different 150 bpclones of melon HPL and AOS. Clone A and B have 65% identity, whereasclone A and C have 57% and B and C have 72% identity in amino acidsequences.

FIG. 5 compares the identities between the partial amino acid sequencesencoded by the 3′ ends of Clones A, B and C from melon and theC-terminal sequences of 13-HPL from guava, pepper and banana and AOSfrom flax, guayule, and Arabidopsis. The C-terminal sequences encoded byClone A and B have 42% identity, whereas clone A and C have 40% and Band C have 49% identity.

FIG. 6 shows a schematic of the two primary enzymatic products of9S-hydroperoxylinolcic acid in the presence of melon 9-HPL:9-oxo-nonanic acid and 3Z-nonenal. Also depicted is the minorisomerization reaction of 3Z-nonenal to 2E-nonenal, that is observed toa small extent using either the purified enzyme or the crude bacteriallysate. Also depicted is the oxidation reaction that occurs with thecrude bacterial lysate, whereby, 3Z-nonenal is oxidized to a mixture ofthree aldehydes, 4-hydroxy- 2E-nonenal (4-HNE), and4-hydroperoxy-2E-nonenal (4-HPNE), and a hemiacetal derivative formedbetween 9-oxo-nonanic acid and 4-hydroperoxy-2E-nonenal (hemiacetal).

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of preferred embodiments of the inventionand the Examples included therein.

Before the present methods are disclosed and described, it is to beunderstood that this invention is not limited to specific methods or toparticular formulations, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only and is not intended to belimiting. As used in the specification and in the claims, “a” can meanone or more, depending upon the context in which it is used.

A. Proteins and Nucleic Acids

The present invention provides a fatty acid lyase and a nucleic acidencoding the lyase. In particular, an isolated fatty acid hydroperoxidelyase is disclosed, wherein the activity of the lyase for9-hydroperoxide substrates is greater than the activity for13-hydroperoxide substrates and wherein K_(m) and V_(max) of the lyasefor 9-hydroperoxylinolenic acid are greater than K_(m) and V_(max) ofthe lyase for 9-hydroperoxylinoleic acid. More particularly, theinvention provides a lyase present in melon (Cucumis melo), but not incucumber (Cucumis sativus), and a nucleic acid encoding such polypeptideor protein. Thus, the lyase has an amino acid sequence present in aprotein isolated from Cucumis melo, but does not have an amino acidsequence in a protein isolated from cucumber (Cucuimis sativus).

The term “protein” refers to a polymer of amino acids and can includefull-length proteins and polypeptides and fragments thereof. In thepresent invention, “lyase” means a protein having at least one lyasefunction. In particular, the term “9-hydroperoxide lyase,” “9-HPL,” and“functional 9-hydroperoxide lyase” mean a lyase protein having at leastone function exhibited by a native 9-hydroperoxide lyasc. For example,9-HPL function can include the catalytic activity of cleaving a fattyacid 9-hydroperoxide into a C-9 aldehyde and a C-9-oxononanoic acid.Additionally, the disclosed lyases can have the followingcharacteristics of native 9-HPL: antigenic determinants, bindingregions, or the like.

The disclosed 9-HPL prefers 9-hydroperoxide substrates rather than13-hydroperoxide substrates but has both 9-HPL and 13-HPL functions. Theterms “13-hydroperoxide lyase,” “13-HPL,” and “functional13-hydroperoxide lyase” refer to a lyase protein having at least onefunction exhibited by a native 13-hydroperoxide lyase. For example,13-HPL function can include the catalytic activity of cleaving a fattyacid 9-hydroperoxide into a C-6 aldehyde and a C-12-ω-oxoacid moiety.Additionally, the disclosed lyases can have the followingcharacteristics of native 13-HPL: antigenic determinants, bindingregions, or the like.

The lyase of the present invention can comprise additional amino acids,such as amino acids linked to the N-terminal end or amino acids linkedto the C-terninal end or amino acids inserted within the lyase sequence,as long as the resulting protein or peptide retains a lyase function,such as the preferred lyase function. Furthermore, the lyase can containvarious mutations in the amino acid sequence compared to the amino acidsequence of a native lyase, so long as at least one lyase function ismaintained. More specifically, the disclosed lyase cleaves9-hydroperoxylinoleic substrates (e.g., (9S, 10E, 12Z)9-hydroperoxyoctadeca- 10,12-dienoic acid), 9-hydroperoxylinolenicsubstrates (e.g., (9S, 10E, 12Z, 15Z)9-hydroperoxyoctadeca-10,12,15-trienoic acid), 13-hydroperoxylinoleicsubstrates (e.g., (9Z, 11E, 13S)13-hydroperoxyoctadeca-9,11-dienoicacid), and 13-hydroperoxylinolenic substrates (e.g., (9Z,11E, 13S, 15Z)13-hydroperoxyoctadeca-9,11,15-trienoic acid). The K_(m) and V_(max) ofthe lyase for 9-hydroperoxylinolenic acid are greater than K_(m) andV_(max) of the lyase for 9-hydroperoxylinoleic acid.

The lyase has a characteristic affinity for various substrates. Thelyase has a greater affinity for 13-hydroperoxide substrates, and theK_(m) of the lyase for 9-hydroperoxide substrates is greater than for13-hydroperoxide substrates. The computed K_(m) is as follows:9-hydroperoxylinolenic acid>9-hydroperoxylinoleicacid>13-hydroperoxylinoleic acid. The K_(m) of the lyase for13-hydroperoxylinoleic acid is approximately the same as the affinityfor 13-hydroperoxylinolenic acid. More specifically, the computed K_(m)for 9-hydroperoxylinoleic acid is approximately 192 μM with 95%confidence limits as 142-242 and is approximately 45-60%, and preferablyapproximately 54%, of the K_(m) of the lyase for 9-hydroperoxylinolenicacid. The computed K_(m) for 13-hydroperoxylinolenic acid isapproximately 50 μM with 95% confidence limits as 41-59 and isapproximately 15-35%, and preferably approximately 26%, of the K_(m) ofthe lyase for 9-hydroperoxylinolenic acid. The computed K_(m) for13-hydroperoxylinolenic acid is approximately 51 μM with 95% confidencelimits as 37-65 and is approximately 15-35%, and preferablyapproximately 27%, of the K_(m) of the lyase for 9-hydroperoxylinolenicacid.

The disclosed lyase cleaves each type of substrate with a characteristicrate. The lyase reacts faster with the 9-hydroperoxide substrates, andthe V_(max) of the lyase for 9-hydroperoxide substrates is greater thanthe V_(max) for 13-hydroperoxide substrates. The rate of cleavage of thevarious substrates by the lyase of the invention, as indicated byV_(max), is as follows:9-hydroperoxylinolenic acid>9-hydroperoxylinoleicacid>13-hydroperoxylinoleic acid. The rate for 13-hydroperoxylinoleicacid is approximately the same as the rate for 13-hydroperoxylinolenicacid. More specifically, V_(max) of the lyase for 9-hydroperoxylinoleicacid is approximately 45-60%, and preferably approximately 55%, of theV_(max) of the lyase for 9-hydroperoxylinolenic acid. V_(max) of thelyase for 13-hydroperoxylinoleic acid is approximately 25-35%, andpreferably approximately 30%, of the V_(max) of the lyase for9-hydroperoxylinolenic acid. V_(max) of the lyase for13-hydroperoxylinolenic acid is approximately 20-30%, and preferablyapproximately 22%, of the V_(max) of the lyase for9-hydropcroxylinolenic acid. By “approximately the same” rate oraffinity is meant that the rate or affinity for one substrate, e.g.,13-hydroperoxylinolenic acid, as expressed as a percentage of the rateor affinity for 9- hydroperoxylinolenic acid, is within 10%, andpreferably within 5%, of a second substrate, e.g.,13-hydroperoxylinoleic acid, also expressed as a percentage of the rateor affinity for 9- hydroperoxylinolenic acid.

The disclosed lyase has a molecular weight of about 45-65 kDa,preferably about 50-60kDa, and even more preferably about 55 kDa. Theoptimal pH for the disclosed lyase is greater than 6, preferably about6.5-8.5, more preferably 7.0-8.0, and even more preferably 7.2-7.6. Theenzyme has approximately 25% of maximal activity at pH 5.0 andapproximately 15% of maximal activity at pH 9.0.

The disclosed lyase is isolated. Isolation of the lyase can occur in avariety of ways. For example, the lyases can be purified, or partiallypurified, from a source, such as Cucumis melo, using standardbiochemical techniques. See, for example, Homostaj and Robinson (1998).Alternatively, the lyase can be synthesized using protein synthesistechniques known in the art or can be recombinantly produced, throughrecombinant DNA technology and the use of genetically engineeredexpression systems. Synthesized or recombinantly produced lyase can betagged with histidines to promote isolation. Thus, a preferred isolationmethod for recombinantly produced lyase is the use of nickel columns,which bind histidine residues. Histidine residues can be added to theamino terminal end of the disclosed lyase to act as a tag for theprotein. The use of histidine tags or other tags is well know to one ofordinary skill in the art.

In one embodiment, the disclosed lyase comprises amino acids unique toCucumis melo, as set forth in FIG. 1, that provide the activity ofcleaving 9-hydroperoxide substrates with greater activity than13-hydroperoxide substrates and that provide the activity of cleaving9-hydropoxylinoleic acid with less than 1.6 times the activity as9-hydroperoxylinolenic acid.

The invention also provides an isolated protein, comprising an aminoacid sequence selected from the group consisting of SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,and SEQ ID NO: 15. The amino acid sequence of SEQ ID NO:15 has beensubmitted to the GenBank database under accession number AF081955.

The invention provides an isolated nucleic acid that encodes thedisclosed lyase. The cDNA of the 9-HPL from Cucumis melo has been clonedand sequenced (SEQ ID NO:8). The amino acid sequence of the proteinencoded by the Cucumis melo cDNA is also disclosed (SEQ ID NO:7). In oneembodiment, the nucleic acid comprises the nucleic acid sequence setforth in SEQ ID NO:8. In another embodiment, the nucleic acid comprisesthe nucleic acid sequence set forth in SEQ ID NO:56. The nucleic acidsequence of SEQ ID NO:56 has been submitted to the GenBank databaseunder accession number AF081955.

Further provided are isolated nucleic acids that encode the proteinhaving an amino acid sequence selected from the group consisting of SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, and SEQ ID NO:7. Recombinant systems include expression systems inboth prokaryotic and eukaryotic cells and include expression of thelyase having the native protein sequence or the lyase having a proteinsequence altered from the native sequence in some way. The melon 9-HPLCDNA was cloned and sequenced and the nucleotide sequence for thefull-length cDNA was determined to be 1446 base pairs (SEQ ID NO:8),which includes a stop codon. The translated sequence encodes a total of481 amino acids residues (SEQ ID NO:7), corresponding to a protein witha calculated molecular weight of about 55,000 Daltons.

As shown in FIG. 1, the derived full length amino acid sequence shows adegree of homology (identity and similarity) to a number of HPLs andallene oxide synthases (AOS). For example, there is a degree of homologybetween the disclosed amino acid sequence and the 13-HPLs of guava,banana, and pepper. There is also homology between the disclosed HPL andAOS-Flax, AOS-Guayule, AOS Arabi, and AOS-Melon. However, FIG. 1 clearlydemonstrates that there are regions for the disclosed lyase that areunique relative to other HPLs and AOSs. Particularly these regions areunique to 9-HPLs and furthermore these regions are unique to Cucumismelo.

Taking into account deletions and insertions, the alignment in FIG. 1and Table 1 reveal that, using the Clustal method with PAM250 residueweight chart available through the MegAlign subprogram of Lasergenc(Dnastar, Madison, Wis.), the melon 9-HPL amino acid sequence has abouta 45.7% similarity with AOS-Flax, about a 46% similarity withAOS-Guayule, about a 48.0% similarity with AOS-Arabi, about a 47%similarity with AOS-Melon, about a 60% similarity with HPL-Guava, abouta 58% similarity with HPL-Banana, and about a 60% similarity withHPL-Pepper.

“Similarity” can include amino acid residues that are either the same orsimilar. Similar amino acids are indicated in Table 2. Despite thesesimilarities, there are uniques regions of the disclosed lyase.Preferred unique regions are set forth in SEQ ID NO:1 (MATPSSSSPE), SEQID NO:2 (ILFDTAKVEKRNILD), SEQ ID NO:3 (RLFLSFLA), SEQ ID NO:4(SISDSMS), SEQ ID NO:5 (LLSDGTPD), and SEQ ID NO:6 (IFSVFEDLVI).Proteins that contain these regions and function as the disclosed lyaseare provided. Particularly preferred embodiments are those that have atleast one of these defined regions set forth in SEQ ID NOs:1-6 thatretain 9-HPL function. More preferred embodiments are those that havc atleast two of these defined regions set forth in SEQ ID NOs: 1-6 presentand that retain 9-HPL function. More preferred embodiments are thosethat have at least three of these defined regions set forth in SEQ IDNOs:1-6 and that retain 9-HPL function. More preferred embodiments arcthose that have at least four of these defined regions set forth in SEQID NOs:1-6 and that retain 9-HPL function. Even more preferredembodiments are those that have at least five of these defined regionsset forth in SEQ ID NOs:1-6 and that retain 9-HPL function. Mostpreferred embodiments are those that have at least six of the regionsset forth in SEQ ID NOs:1-6 and that retain 9-HPL function.

TABLE 1 Percent Similarity 1 2 3 4 5 6 7 8 Percent 1 59.2 56.5 59.4 36.237.2 34.9 44.7 1 AOS-Flax Divergence 2 33.6 57.0 55.8 42.1 46.1 43.855.5 2 AOS-Guayule 3 40.6 39.5 56.8 37.8 38.9 36.7 47.8 3 AOS-Arabi 438.3 36.6 40.4 35.1 37.6 33.0 45.8 4 AOS-Melon 5 58.9 56.6 60.7 60.960.5 67.3 42.3 5 HPL-Guava 6 56.1 55.4 57.2 56.2 39.6 58.4 46.4 6HPL-Banana 7 59.2 58.7 60.4 61.5 32.4 45.0 44.3 7 HPL-Pepper 8 47.1 46.447.5 47.2 59.6 57.5 59.3 8 HPL-Melon 1 2 3 4 5 6 7 8

It is understood that the disclosed lyase includes functional variants.These variants are produced by making amino acid substitutions,deletions, and insertions, as well as post-translational modifications.Such variations may arise naturally as allelic variations (e.g., due togenetic polymorphism) or may be produced by human intervention (e.g., bymutagenesis of cloned DNA sequences), such as induced point, deletion,insertion and substitution mutants. These modifications can result inchanges in the amino acid sequence, provide silent mutations, modify arestriction site, or provide other specific mutations.

Amino acid sequence modifications fall into one or more of threeclasses: substitutional, insertional or deletional variants. Insertionsinclude amino and/or carboxyl termninal fusions as well as intrasequenceinsertions of single or multiple amino acid residues. Insertionsordinarily will be smaller insertions than those of amino or carboxylterminal fusions, for example, on the order of one to four residues.Deletions are characterized by the removal of one or more amino acidresidues from the protein sequence. Typically, no more than about from 2to 6 residues are deleted at any one site within the protein molecule.These variants ordinarily are prepared by site specific mutagenesis ofnucleotides in the DNA encoding the protein, thereby producing DNAencoding the variant, and thereafter expressing the DNA in recombinantcell culture. Techniques for making substitution mutations atpredetermined sites in DNA having a known sequence are well known, forexample M13 primer mutagenesis and PCR mutagenesis. Amino acidsubstitutions are typically of single residues but may include multiplesubstitutions at different positions; insertions usually will be on theorder of about from 1 to 10 amino acid residues but can be more; anddeletions will range about from 1 to 30 residues, but can be more.Deletions or insertions preferably are made in adjacent pairs, i.e. adeletion of 2 residues or insertion of 2 residues. Substitutions,deletions, insertions or any combination thereof may bc combined toarrive at a final construct. The mutations must not place the sequenceout of reading frame and preferably will not create complementaryregions that could produce secondary mRNA structure. Substitutionalvariants are those in which at least one residue has been removed and adifferent residue inserted in its place. Such substitutions generallyare made in accordance with Table 2 and are referred to as conservativesubstitutions.

TABLE 2 Amino Acid Substitutions Original Residue ExemplarySubstitutions Ala ser Arg lys Asn gln Asp glu Cys ser Gln asn Glu aspAla ser Gly pro His gln Ile leu; val Leu ile; val Lys arg; gln Met leu;ile Phe met; leu; tyr Ser thr Thr ser Trp tyr Tyr trp; phe Val ile; leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those in Table2, i.e., selecting residues that differ more significantly in theireffect on maintaining (a) the structure of the polypeptide backbone inthe area of the substitution, for example as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site or (c) the bulk of the side chain. The substitutions whichin general are expected to produce the greatest changes in the proteinproperties will be those in which (a) a hydrophilic residue, e.g. serylor threonyl, is substituted for (or by) a hydrophobic residue, e.g.leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g., lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) one not having a side chain,e.g., glycine, in this case, (e) by increasing the number of sites forsulfation and/or glycosylation.

Substitutional or deletional mutagenesis can be employed to insert sitesfor N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr).Deletions of cysteine or other labile residues also may be desirable.Deletions or substitutions of potential proteolysis sites, e.g. Arg, isaccomplished for example by deleting one of the basic residues orsubstituting one by glutaminyl or histidyl residues.

Certain post-translational derivatizations are the result of the actionof recombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and asparyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Otherpost-translational modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, methylation of the o-amino groups of lysine, arginine, andhistidine side chains (Creighton,1983), acetylation of the N-tenninalamine and, in some instances, amidation of the C-terminal carboxyl.

In all mutational events, it is understood that the controlling aspectof the mutation is the function that the subsequent protein possesses.The most preferred mutations are those that do not detectably change the9-HPL function. For example as described above the disclosed lyase hasvery specific kinetic characteristics and preferred mutations would bethose that for example produce mutated 9-HPLs that preferentially cleave9-hydroperoxide substrates.

There are numerous assays for determining the relative function of thedisclosed lyases, including, for example, HPLC analysis,spcctrophotometric analysis, gas chromatographic analysis, and gaschromatography with mass spectrometric analysis.

It is also understood that mutational events may at times includemutations that alter the activity in a defined way, for example, byincreasing the V_(max) of cleavage of 9-hydroperoxide substrates. Shouldthese types of mutations be desired, close analysis of the reactionrates and function of the mutated proteins will allow isolation ofmutant lyases that either function better or worse than native lyases.Preferred mutations are those that increase the activity of the lyasefor cleavage of 9-hydroperoxide substrates.

It is also understood that there is degeneracy in the relationshipbetween nucleic acids and proteins so that there can be multiple nucleicacid codons for a given protein sequence. Thus, the melon cDNA, whilenot having the same sequence as the DNA isolated from Cucumis melo,encodes the same amino acid sequence of the lyase isolated from Cucumismelo. In addition, there are numerous reasons one may wish to alter thesequence of the Cucumis melo CDNA while maintaining the unique coding ofthe Cucunis melo protein. For example, one may wish to insert or removespecific nucleic acid restriction enzyme sites contained or desired inthe CDNA.

Particularly preferred embodiments incorporate both the functionalvariants incorporating non-conserved amino acids described above incombination with the unique regions set forth in SEQ ID NOs:1-6. Mostpreferred is the functional 9-HPL isolated from Cucumis melo having thesequence set forth in SEQ ID NO:7.

Also disclosed are nucleic acid sequences that encode the proteinsdisclosed herein. These nucleic acids would include those that encode aprotein possessing at least one of the unique amino acid sequencesdisclosed in SEQ ID NOs:1-6. This would include as discussed above alldegenerate sequences to the nucleic acids encoding these proteins. Oneembodiment is the nucleic acid representing the CDNA isolated fromCucumis melo, as set forth in SEQ ID NO:8.

Also disclosed are isolated nucleic acids, which specifically hybridizewith the nucleic acid of SEQ ID NO:8 under stringent conditions ofhybridization. Preferably the nucleic acids that hybridize with thenucleic acid of SEQ ID NO:8 under stringent conditions do not hybridizeat the stringent conditions with a nucleic acid encoding a lyase presentin Cucumis sativus. Most preferably the isolated nucleic acid encodes aprotein that has a 9-HPL function.

“Stringent conditions” refers to the washing conditions used in ahybridization protocol or in a primer/template hybridization in a PCRreaction. In general, these conditions should be a combination oftemperatures and salt concentrations for washing chosen so that thedenaturation temperature is approximately 5-20° C. below the calculatedT_(m) (melting/denaturation temperature) of the hybrid under study. Thetemperature and salt conditions are readily determined empirically inpreliminary experiments in which samples of reference nucleic acid arehybridized to the primer nucleic acid of interest and then amplifiedunder conditions of different stringencies. The stringency conditionsare readily tested and the parameters altered are readily apparent toone skilled in the art. For example, MgCl₂ concentrations used in PCRbuffer can be altered to increase the specificity with which the primerbinds to the template, but the concentration range of this compound usedin hybridization reactions is narrow, and, therefore, the properstringency level is easily determined. For example, hybridizations witholigonucleotide probes 18 nucleotides in length can be done at 5-10° C.below the estimated T_(m) in 6X SSPE, then washed at the sametemperature in 2X SSPE. The T_(m) of such an oligonuclcotide can beestimated by allowing 2° C. for each A or T nucleotide, and 4° C. foreach G or C. An 18 nucleotide probe of 50% G+C would, therefore, have anapproximate T_(m) of 54° C. Likewise, the starting salt concentration ofan 18 nucleotide primer or probe would be about 100-200 mM. Thus,stringent conditions for such an 18 nucleotide primer or probe would bea T_(m) of about 54° C. and a starting salt concentration of about 150mM and modified accordingly by preliminary experiments. T_(m) values canalso be calculated for a variety of conditions utilizing commerciallyavailable computer software (e.g., OLIGO®).

The present invention further provides an isolated nucleic acid whichspecifically hybridizes with the nucleic acid encoding the amino acidsequence of melon 9-HPL, as set forth in SEQ ID NO:7, under stringentconditions of hybridization. Preferably, the isolated nucleic acid doesnot hybridize at the stringent conditions to a nucleic acid set encodinga lyase present in Cucumis sativus. Most preferably the isolated nucleicacid encodes a protein that has a 9-HPL function.

Preferably, the isolated nucleic acid of the invention has at least 99,98, 97, 95, 90, 85, 80, 75, or 70% complementarity with the sequence towhich it hybridizes. More preferred embodiments are isolated nucleicacids that have at least 90% complementarity with the sequence to whichit hybridizes. More preferred embodiments are isolated nucleic acidsthat have at least 80% complementarity with the sequence to which ithybridizes. More preferred embodiments are isolated nucleic acids thathave at least 70% complementarity with the sequence to which ithybridizes. The percent complementarity can be based preferably on anucleotide-by-nucleotide comparison of the two strands. Specific methodsof determining complementarity are well known in the art (e.g., theClustal, Jotun Hein, WilburLipman, Martinez Needleman-Wunsch,Lipman-Pearson, and Dotplot methods). A skilled artisan, therefore,would understand the meaning of the term and would know how to determinecomplementarity between two sequences.

The nucleic acid can also be a probe or a primer, for example, to detector amplify target nucleic acids. Typically, a unique nucleic acid usefulas a primer or probe will be at least about 20 to about 25 nucleotidesin length, depending upon the specific nucleotide content of thesequence. Additionally, fragments can be, for example, at least about30, 40, 50, 75, 100, 200, 400, or any number in between in nucleotidelength. Alternatively, a full length sequence or a sequence that islonger than a full length sequence can be used.

B. Vectors

The invention provides a vector, comprising the nucleic acid of theinvention. The present invention also provides vectors comprising anucleic acid that encodes a 9-hydroperoxide lyase, including, forexample, a lyase having an amino acid sequence present in a proteinisolated from Cucumis melo. More specifically, the vector can be aplasmid. Even more specifically, the vector can comprise a promoterfunctionally linked to one of the nucleic acids of the presentinvention.

“Vector” means any carrier containing exogenous DNA. Thus, vectors areagents that transport the exogenous nucleic acid into a cell withoutdegradation and include a promoter yielding expression of the nucleicacid in the cells into which it is delivered. “Vectors” include but arenot limited to plasmids, viral nucleic acids, viruses, phage nucleicacids, phages, cosmids, and artificial chromosomes. A variety ofprokaryotic and eukaryotic expression vectors suitable for expression ofthe functional lyase of the invention can be produced. Such expressionvectors include, for example, pET, pET3d, pCR2.1, pBAD, pUC, and yeastvectors. The vectors can express the described lyase, for example, in avariety of in vivo and in vitro situations.

Viral vectors include adenovirus, adeno-associated virus, herpes virus,vaccinia virus, polio virus, AIDS virus, neuronal trophic virus, Sindbisand other RNA viruses, including these viruses with the HIV backbone.Also preferred are any viral families which share the properties ofthese viruses which make them suitable for use as vectors. Retroviralvectors, which are described in Verma (1985), include Murine MaloneyLeukemia virus, MMLV, and retroviruses that express the desirableproperties of MMLV as a vector. Typically, viral vectors contain,nonstructural early genes, structural late genes, an RNA polymerase IIItranscript, inverted terminal repeats necessary for replication andencapsidation, and promoters to control the transcription andreplication of the viral genome. When engineered as vectors, virusestypically have one or more of the early genes removed and a gene orgene/promotor cassette is inserted into the viral genome in place of theremoved viral DNA.

A “promoter” is generally a sequence or sequences of DNA that functionwhen in a relatively fixed location in regard to the transcription startsite. A “promoter” contains core elements required for basic interactionof RNA polymerase and transcription factors and may contain upstreamelements and response elements.

“Enhancer” generally refers to a sequence of DNA that functions at nofixed distance from the transcription start site and can be either 5′(Laimins, 1981) or 3′ (Lusky et al., 1983) to the transcription unit.Furthermore, enhancers can be within an intron (Banerji et al., 1983) aswell as within the coding sequence itself (Osborne et al., 1984). Theyare usually between 10 and 300 bp in length, and they function in cis.Enhancers function to increase transcription from nearby promoters.Enhancers, like promoters, also often contain response elements thatmediate the regulation of transcription. Enhancers often determine theregulation of expression. It is preferred that the promoter and/orenhancer region act as a constitutive promoter and/or enhancer tomaximize expression of the region of the transcription unit to betranscribed.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human or nucleated cells) may also contain sequencesnecessary for the termination of transcription which may affect mRNAexpression. These regions are transcribed as polyadenylated segments inthe untranslated portion of the mRNA encoding tissue factor protein. The3′ untranslated regions also include transcription termination sites. Itis preferred that the transcription unit also contain a polyadenylationregion. One benefit of this region is that it increases the likelihoodthat the transcribed unit will be processed and transported like mRNA.The identification and use of polyadenylation signals in expressionconstricts is well established. It is preferred that homologouspolyadenylation signals be used in the transgene constricts.

The vector can include nucleic acid sequence encoding a marker product.This marker product is used to determine if the gene has been deliveredto the cell and once delivered is being expressed. Preferred markergenes are the E. Coli lacZ gene which encodes β-galactosidasc and greenfluorescent protein.

In some embodiments the marker may be a selectable marker. When suchselectable markers are successfully transferred into a host cell, thetransformed host cell can survive if placed under selective pressure.There are two widely used distinct categories of selective regimes. Thefirst category is based on a cell's metabolism and the use of a mutantcell line which lacks the ability to grow independent of a supplementedmedia. The second category is dominant selection which refers to aselection scheme used in any cell type and does not require the use of amutant cell line. These schemes typically use a drug to arrest growth ofa host cell. Those cells which have a novel gene would express a proteinconveying drug resistance and would survive the selection. Examples ofsuch dominant selection use the drugs neomycin, (Southern andBerg,1982), mycophenolic acid, (Mulligan and Berg, 1980) or hygromycin(Sugden et al., 1985).

Also disclosed are cells that containing an exogenous nucleic acidcomprising the nucleic acid encoding the lyase or protein of the presentinvention. A preferred cell is a prokaryotic cell. Particularlypreferred prokaryotic cells are Escherichia coli cell, a Bacillus cell,and a Streptomyces cell. These bacteria have the ability to secreterecombinant proteins, thus, avoiding the need for lysing the cells toisolate the protein.

Another preferred cell type containing an exogenous nucleic acidcomprising the nucleic acid encoding the lyase or protein of the presentinvention is a eukaryotic cell. Particularly preferred eukaryotic cellsare a yeast cell, a plant cell, and an insect cell. For example, Pichiapastoris or Saccharomyces cerevisiae can be used as an expressionsystem. Appropriate means for transfection of the cells with theexogensous nucleic acid, including viral vectors, chemicaltransfectants, or physico-mechanical methods such as electroporation anddirect diffusion of DNA, are well known in the art. See, for example,Wolff et al. (1990) and Wolff (1991), which are incorporated herein intheir entirety by reference. The transfected cells can be used as amethod of expressing the proteins and lyases of the present invention.

Many different strategies can be used to optimize expression of theprotein or lyase of the present invention. Different enhancers areselected based on the host cell type, vector, and promoter. For example,isopropyl β-D-thiogalactopyranoside (IPTG) can be used as an inducer ofthe P_(lac) promoter and derivatives of the P_(lac) promoter when E.coli is the host cell. Inducer concentrations of IPTG range between 0-1mM. Alternatively, a pBAD vector with a promoter that is induced byL-arabinose can be used in E. coli. Host cell type, vector, promoter,induction times, media compositions, temperature, cofactors, cultivationconditions, and cultivation times can be altered to optimize expression.Furthermore, the addition of a precursor of prosthetic groups like heme(including, for example, δ-aminolevulinic acid) can be used to optimizeexpression.

C. Methods of using the Compositions

Disclosed is a method of cleaving a (9S, 10E, 12Z)9-hydroperoxyoctadeca-10,12-dienoic acid or (9S, 10E, 12Z, 15Z)9-hydroperoxyoctadeca-10,12,15-trienoic acid into a C9-aldehyde and a(C9-oxononanoic acid, comprising the steps of contacting the disclosedlyase with the (9S, 10E, 12Z) 9-hydroperoxyoctadeca-10,12-dienoic acidor (9S, 10E, 12Z, 15Z)9-hydroperoxyoctadeca-10,12,15-trienoic acid. When(9S, 10E, 12Z) 9-hydroperoxyoctadeca- 10,12-dienoic acid is thesubstrate, the C9-aldehyde is 3Z-nonenal. When (9S, 10E, 12Z, 15Z)9-hydropcroxyoctadeca-10,12,15-trienoic acid is the substrate, theC9-aldehyde is 3Z, 6Z-nonadienal.

Also disclosed are methods of cleaving (9Z, 11E, 13S)13-hydroperoxyoctadeca-9,11-dienoic acid or (9Z, 11E, 13S, 15Z)13-hydroperoxyoctadeca-9,11,15,-trienoic acid into a C6- aldehyde and aC12-oxocarboxylic acid, comprising contacting the disclosed lyase withthe 13-hydroperoxyoctadeca-9,11-dienoic acid or13-hydroperoxyoctadeca-9,11,15-trienoic acid.

Also disclosed are methods of preparing 3-(Z)-nonenal,(3Z,6Z)-nonadienal, 2-(E)-nonenal, (2E,6Z)-nonadienal, or theircorresponding alcohols from (9S, 10E, 12Z) 9-hydroperoxyoctadeca-10,12-dienoic acid or (9S, 10E, 12Z, 15Z)9-hydroperoxyoctadeca-10,12,15-trienoic acid, comprising the steps of contacting the (9S, 10E,12Z) 9-hydroperoxyoctadeca- 10,12-dienoic acid or (9S, 10E, 12Z,15Z)9-hydroperoxyoctadeca- 10,12,15-trienoic acid with the disclosed9-HPL, thereby converting the (9S, 10E, 12Z)9-hydroperoxyoctadeca-10,12-dienoic acid into 3-(Z)-nonenal or the (9S,10E, 12Z, 15Z)9-hydroperoxyoctadeca-10,12,15-trienoic acid into(3Z,6Z)-nonadienal; and recovering the 3-(Z)-nonenal or(3Z,6Z)-nonadienal; rcducing the 3-(Z)-nonenal into 3-(Z)-nonenol or the(3Z,6Z)-nonadienal into (3Z,6Z)-nonadienol and recovering the3-(Z)-nonenol or (3Z,6Z)-nonadienol; or isomerizing the 3-(Z)-noncnal or(3Z,6Z)-nonadienal under temperature and pH conditions effective toobtain 2-(E)-nonenal or (2E,6Z)-nonadienal and either recovering theformed 2-(E)-nonenal or (2E,6Z)-nonadienal or reducing the 2-(E)-nonenalto 2-(E)-nonenol or the (2E,6Z)-nonadienal to (2E,6Z)-nonadienol andrecovering the 2-(E)-nonenol or (2E,6Z)-nonadienol from the medium. Thereducing step is preferentially carried out using an enzyme catalyzedreduction (e.g., using alcohol dehydrogenase) mediated by yeast usingtechniques known in the art. See,for example, EP 0 597 069 B 1, which isincorporated herein in its entirety by reference. The isomerization stepcan be optimized by using an enzymatic procedure. The isomerization canbe catalyzed by an isomerase or by a non-enzymatic isomerization factor.For example, the isomerase can be a 3Z:2E-enal isomerase. See, e.g.,Noordermeer et al. (1999), which is incorporated herein in its entiretyby reference.

Also disclosed are methods of preparing n-hexanal, 3-(Z)-hexen-1-al,2-(E)-hexen-1-al, or their corresponding alcohols from (9Z, 11E, 13S)13-hydroperoxyoctadeca-9,11-dienoic acid or (9Z, 11E, 13S, 15Z)13-hydroperoxyoctadeca-9,11,15,-trienoic acid, comprising the steps ofcontacting the (9Z, 11E, 13S) 13-hydroperoxyoctadeca-9,11-dienoic acidor (9Z, 11E, 13S, 15Z) 13-hydroperoxyoctadeca-9,11,15,-trienoic acidwith the disclosed 9-HPLs, thereby converting the (9Z, 11E, 13S)13-hydroperoxyoctadeca-9,11-dienoic acid into n-hexanal or the (9Z, 11E,13S, 15Z) 13-hydroperoxyoctadeca-9,11,15,-trienoic acid into3-(Z)-hexen-1-al; and either recovering the n-hexanal or3-(Z)-hexen-1-al; reducing the n-hexanal into n-hexanol or the3-(Z)-hexen-1-al into 3-(Z)-hexen-1-ol and recovering the hexanol or3-(Z)-hexen-1-ol; or isomerizing the 3-(Z)-hexel-1-al under temperatureand pH conditions effective to obtain 2-(E)-hexen-1-al and eitherrecovering the formed 2-(E)-hexcn-1-al or reducing the 2-(E)-hexen-1-alto 2-(E)-hexen-1-ol and recovering the 2-(E)-hexen-1-ol from the medium.The reducing step is preferentially carried out using the enzymecatalyzed reduction described above, and the isomerization step can beoptimized using the enzymatic procedure described above.

The present invention is more particularly described in the followingexamples which are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art.

EXAMPLES Example 1

Cloning of partial cDNAs of melon lyases, including 9-hydroperoxidelyase.

A homology-based cloning method was used to isolate muskmelon (Cucumismelo). Generally, the melon mRNA was prepared, reverse transcriptase wasused to convert melon mRNA to CDNA. This CDNA was the substrate for thepolymerase chain reaction (RT-PCR) using degenerate primers designed tomatch consensus sequences in the cytochrome P450 family 74 (CYP74). ThisPCR provided the partial cDNA clones having sequence homology to theCYP74 gene family. The partial clones were extended by 3′-RACE (RapidAmplification of cDNA Ends) and 5′-RACE reactions, which gave thecomplete cDNA (i.e., the full complement of mRNA) for each partialclone. The full length cDNA(s) were cloned by PCR, and expressed in E.coli. The catalytic activities of the E. coli expressed product wascharacterized using 9-hydroperoxy and 13-hydroperoxy fatty acids assubstrates.

A. Preparation of melon RNA

The starting material was Cantaloupe melon (“muskmelon”), Cucumis melo,of the variety, Caravelle (Asgrow, Tex.). A TRI REAGENT kit (MolecularResearch Center, Cincinnati, Ohio) was used to isolate the total RNA.Total RNA was prepared from 20g of immature melon fruit. 400μg of totalRNA were obtained. An mRNA purification kit (Pharmacia Biotech,Piscataway, N.J.) was used to purify the mRNA from total RNA. The kitprovides oligo(dT)-cellulose spin columns for the affinity purificationof polyadenylated RNA. The manufacturer's protocol was followed. 3.7 μgof mRNA was isolated from 400 μg of total RNA.

B. RT-PCR cloning using degenerate primers based on conserved CYP74sequences First strand cDNA was synthesized from total RNA orpoly(A)+RNA using an oligo-d(T)-adaptor. The reverse transcriptasereaction contained 80 pmoles of oligo-dT adaptor (SEQ ID NO:49, A 1678,5′-ATG AAT TCG GTA CCC GGG ATC CTT TTT TTT TTT TTT TTT-3′ or SEQ IDNO:50, A 1677, 5′-ATG AAT TCG GTA CCC GGG ATC-3′), 10 μl of 5x firststrand buffer (GibcoBRL, Rockville, Md., 1 mM DTT, 1 mM for each dNTP,50 units RNAsin, 400 U MMV-RT, and H₂O to a final reaction volume of 50μl. This RT reaction mixture was incubated at 37′ for one hour. Thefirst strand cDNA was used directly in PCR reactions without furtherpurification. The PCR reaction contained 20-100 ng of melon cDNAtemplate, 200 μM of each dNTP, 10 mM Tris HCI pH 8.3, 50 mM KCI, 3mMMgCl₂, 20 pmoles of upstream primer (GGTGAGTTGCTNTGYGGNTAYCA (SEQ IDNO:16), GGTGAGTTGCTNTGYGGNTA (SEQ ID NO:17), or TACTGGTCNAAYGGNCCNSARAC(SEQ ID NO:19)) and 20 pmoles of downstream primer (TGGTCNAAYGGNCCRGAGAC(SEQ ID NO:18), AAYAARCARTGYGCNGCTAAGGAC (SEQ ID NO:20), orAARCARTGYGCNGCTAAGGAC (SEQ ID NO:21) (See FIGS. 2 and 3). The PCRreaction further contained 1.25 units of enzyme and H₂O to a finalreaction volume of 50 μl. The cDNA template was added when the reactiontemperature was 80° C. The reaction cycle parameters were 94° C. for 2minutes (cycle 1 only); 57° to 62° C. for 1 minute, 72° C. for oneminute, 94° for one minutc (typically 30 cycles); and 72° C. for 10minutes (last cycle). The reaction conditions were the same for allreactions, but two different DNA polymerases were used: (1) AmpliTaq DNApolymerase (PE Applied Biosystems, Focter City, Calif.) and (2) AdvanTaq(Advantage cDNA Polymerase Mix (Clontech, Palo Alto, Calif.)).

i. Amplification of the 150 bp cDNA fragment

A single cycle PCR was perfonned using melon cDNA as the template. Theupstream degenerate primer (SEQ ID NO:16, primer 1A, FIGS. 2 and 3) wasused with the downstream degenerate primer (SEQ ID NO:18, primer 2,FIGS. 2 and 3), but no band was obtained in this first PCR. Thus, asecond PCR was performed using 0.1 μl of the first round PCR reactionproducts as template, and using the upstream degenerate primer 1 B (SEQID NO:17, FIGS. 2 and 3) as a nested upstream primer. This second PCRproduced a product that migrated as a unique band (150 bp) in an agarosegel. The 150 bp PCR product is comparable in size to the expected Cyp74gene family product.

The 150 bp product was subcloned into a vector (pCR2.1 by Invitrogen,Carlsbad, Calif.), and about 50 clones were sequenced. Three differentP450-related sequences were obtained (FIG. 4), and these were designatedpartial Clone A (SEQ ID NO:28), Clone B (SEQ ID NO:29), and Clone C (SEQID NO:30). Partial clones A and B have 65% identity homology; partialclones A and C have 57% identity homology; and partial clones B and Chave 72% identity homology.

ii. Amplification of the 70 bp cDNA fragment

The single cycle PCR was performed using melon cDNA as template. Theupstream degencrate primer (SEQ ID NO:18, Primer 2, FIGS. 2 and 3) wasused with a downstream degenerate primer (SEQ ID NO:20, primer 4A, FIGS.2 and 3). No product band was observed in an agarose gel. Thus, a secondPCR was performed using 0.1 ml of the first PCR as template. Thedownstream degenerate primer, primer 4B, (SEQ ID NO:21, FIGS. 2 and 3)was used as a nested upstream primer. This second PCR produced a productthat migrated as a unique band of about 70 bp in an agarose gel. This iscomparable in size to the expected product. As the size of this 70 bpband was hard to determine exactly on agarose gels, individual clones(48 clones) were sized by polyacrylamide gel electrophoresis (PAGE) on a10% gel, using a 10 bp DNA ladder for calibration. The PAGE indicatedthat a complex mixture of products (60-90 bps) was amplified. Twelveclones close to the predicted size were sequenced. One of these clonesencoded a P450-like sequence. This partial clone represented a differentregion of the 150bp partial clone B.

Example 2

Generation of full length clones using 3′-RACE and 5′-RACE derivedprimers

The 3′-RACE (3′- Rapid Amplification of cDNA Ends) method utilizes adegenerate upstream primer for PCR, and a downstream primer based on theadaptor sequence at the 5′-end of the primer used in the reversetranscriptase-catalyzed synthesis of the cDNA. The cDNA was prepared asdescribed in Example 1.

The Marathon cDNA Amplification Kit (Clontech)was used for the 5′-RACE(5′-Rapid Amplification of cDNA Ends). This procedure is designed toconvert mRNA (1 μg) into double stranded cDNA and tag the cDNA ends withan adaptor sequence cassette. The protocol followed was that of themanufacturer.

A. 3′-RACE

The cDNA was prepared as described above. Three different preparationsof total RNA were used: (1) from the mix of juicy flesh and hard rind ofthe melon, (2) from the hard rind of the melon, (3) from the juicy fleshof the melon. A gene-specific upstream primer of clone A(5′-GGTTATCAGCCGCTGGTGATG-3′ (SEQ ID NO:34) or5′-ATGAACCGGAGGCGTTTAATCCG-3′ (SEQ ID NO:35)), B(5′-ACAGAGCGGACGAGTTCGTACCT3′ (SEQ ID NO:36)) or C(5′-AGGATTCGGAGAAGTTCGTGGGC-3′ (SEQ ID NO:37)) was used with adownstream primer based on the oligo dT-adaptor sequence (SEQ ID NO:49and 50).

To isolate the full length clones of clone B and C, the gene specificprimers for clone B (SEQ ID NO:36) and for clone C (SEQ ID NO:37) andthe primer based on the adaptor sequence of the oligo-dT primer (SEQ IDNO:50) were used. The PCR was primed with the cDNA template obtainedfrom the RNA isolated from the mix ofjuicy flesh and hard rind of themelon. PCR reactions using these primers produced a 350 bp (clone B)product and a 550 bp product (clone C) that migrated as unique bands onan agarose gel.

These 350 and 550 bp PCR products were comparable in size to theexpected product from the amplification of the 3′-end of the AOS and13-HPL cDNAs. These products were subcloned into pCR2.1 and sequenced.

To isolate the full length clone of clone A, the PCR was primed with thejuicy flesh or hard rind melon cDNA template. The gene-specific upstreamprimer for clone A (SEQ ID NO:34 or SEQ ID NO:35) and a downstreamprimer based on the oligo dT-adaptor sequence (SEQ ID NO:50) were usedfor amplification. When the PCR reaction was primed with the hard rindmelon cDNA, no PCR product was obtained as determined by agarose gelelectrophoresis. When the PCR reaction was primed with the juicy fleshmelon cDNA, however, two products were obtained that migrated as uniquebands on an agarose gel. The product produced with the primer having thenucleotide sequence of SEQ ID NO:34 was 450 bp and the product producedwith the primer having the nucleotide sequence of SEQ ID NO:35 was 400bp. The difference in size of these two PCR products (50 bp) matched theexpected distance between the two upstream primers corresponding to SEQID NO:34 and SEQ ID NO:35.

The 400 and 450 bp PCR products produced from primers derived from cloneA were comparable in size to the expected product from the 3′-end of theAOS and 13-HPL cDNAs. These products were subcloned into pCR2.1 andsequenced.

FIG. 5 compares the identities between the C-terminal sequences of theamino acid sequences encoded by Clones A, B and C from melon and theC-terminal sequences of 13-HPL from guava, pepper and banana and AOSfrom flax, guayule, and Arabidopsis. This alignment shows that clone Ahas the most homology with the 13-HPL sequences. Clone B and C have morehomology with AOS than with 13-HPL. Clone B is more like AOS than cloneC, and, therefore, clone C is the most divergent from either the AOS or13-HPL,.

B. 5′-RACE

Total RNA was prepared from the juicy flesh melon as described above.The cDNA synthesis for 5′-RACE was accomplished using the Clonetechprocedure (Marathon cDNA Amplification Kit). The protocol followed wasthat of the manufacturer. 1μg of the mRNA from immature melon fruit wasused. A first PCR was performed with melon cDNA as template which wastagged with the Marathon adaptor sequence at the 5′ and 3′-ends. Theupstream primer API was used with a gene-specific downstream primer(5′-CCG TCA GCA CCA CCA AAT CCT TC-3′ (SEQ ID NO:39)) for clone A, 5′-CTG AAC CGA CCG CGA CTG TGT-3′ (SEQ ID NO:41) for clone B, and 5′-TCCGCG TCG GCT CCA CTG TC-3′ (SEQ ID NO:43) for clone C). A product, whichmigrated as a diffuse smeared band on an agarose gel, was obtained inthis first PCR for each clone. A second PCR was performed using 0.05 μlof the first PCR products as template (a 50 μl PCR reaction). Theupstream primer was the adaptor AP2 (Marathon cDNA Amplification Kit)and the downstream gene-specific primer was either 5′-GAA CAG ATA ATCCAG CAG GGC-3′ (SEQ ID NO:40) for clone A, 5′-TCG CCC GTG AAC CGA TCAGGT A-3′ (SEQ ID NO:42) for clone B, or 5′-TCT CCC ACG AAC CTA TCG CCCA-3′ (SEQ ID NO:44) for clone C. This second PCR produced a 1000 bpproduct for clone A, a 1400 bp product for clone B, and a 1200 bpproduct for clone C. The 1000 bp, 1400 bp and 1200 bp PCR products arecomparable in size to the expected product based on the size of the AOSand 13-HPL cDNAs. These products were subcloned into a vector (pCR2.1,Invitrogen) and sequenced.

After sequencing the 5′ and 3′-RACE products of clones B and C,gene-specific primers were synthesized corresponding to the putativestart of the coding sequence and at the stop codon. For Clone B, NcoIand EcoRI restriction sites (unique sites) were incorporated at the 5′and 3′-ends respectively using the following primers 5′-GCC ATG GCC TCCATT GTC ATT CCT TC-3′ (SEQ ID NO:45) (NcoI site in bold and bold ATGcodes for MET) (5′-up) and 5′- GGA ATT CTT AGT GAT GGT GAT GGT GAT GGAAAC TTG CTT TCT TTA G-3′ (SEQ ID NO:46) (EcoRI site in bold and GT codonrepresents stop codon) (3′-down).

For clone C, unique NdeI and ClaI restriction sites were incorporated atthe 5′ and 3′-ends respectively, using the following primers 5′-GCA TATGGC TAC TCC TTC TTC CTC CTC-3′(SEQ ID NO:47) (NdeI site in bold and boldATG codes for MET) (5′-up) and 5′-CAT CGA TTT AGT GAT GGT GAT GGT GATGAT TAG TCA TTA GCT TTA A-3′ (SEQ ID NO:48) (ClaI site in bold and AGTis a stop codon) (3′-down). A NcoI site is present in the codingsequence.

The PCR reaction was primed with the melon cDNA prepared from 1 μg ofmRNA (as described above) and using either the primer having thenucleotide sequence of SEQ ID NO:45 and the primer having the nucleotidesequence of SEQ ID NO:46 or the primer having the nucleotide sequence ofSEQ ID NO:47 and the primer having the nucleotide sequence of SEQ IDNO:48 as primers. The annealing temperature for these reactions was 60°C., and the Advantage cDNA polymerase mix by Clontech was used. A 1.6 kbproduct for clone B and a 1.4 kb product for clone C were amplified.Each of these products was subcloned into a vector (pCR2.1) andsequenced. The nucleotide sequence of clone B is provided as SEQ IDNO:51, and the nucleotide sequence of clone C is provided as SEQ IDNO:7.

The predicted amino acid sequences encoded by the 1.6kb product of cloneB SEQ ID NO:51 (designated melon AOS in FIG. 1 and having amino acidsequence SEQ ID NO:52) and the 1.4 kb product of clone C (designatedmelon HPL in FIG. 1 and having SEQ ID NO:7) were compared to the aminoacid sequences of AOS from flax (SEQ ID NO:53), guayule (SEQ ID NO:54),and arabidopsis (SEQ ID NO:55) and the amino acid sequence of 13-HPLfrom guava (SEQ ID NO:38), banana (SEQ ID NO:33) and pepper (SEQ IDNO:32). Note that the start of the sequences (encoded by the 5′ ends)contain considerable variations in length and in amino acid sequencebefore all the sequences converge and begin to show close relatedness.Clone B has a very long 5′-end, which accounts for the longer 5′-RACEproduct compared to Clone C with a comparatively short 5′ end. Bysequence comparison of the available 3′-end, Clone A most resembled theknown 13-HPL enzymes. Clone B is a melon AOS. Clone C is a melon9-hydroperoxide lyase.

Example 3

Expression in E. coli.

Clone B cDNA in pCR2.1 was cut with NcoI and EcoRI and subcloned intothe expression vector plasmid pET3d (digested also with NcoI and EcoRI).Clone C cDNA in pCR2.1 was cut with NdeI and ClaI and subcloned into theexpression vector plasmid pET3b (digested also with NdeI and ClaI). Thetwo different constructs were used to transform E. Coli, strainBL21(DE3) to express the gene product of clones B and C. Theseconstructs gave bacterial expression of the native plant sequences withno additional amino acids or other modification of the 5′-ends.

For expression, the transformed BL21 cells were cultured overnight at37° C. and 280 rpm in LB medium (3 ml, prepared by dissolving tryptone(10 g), yeast extract (5 g), and NaCl (10 g) in 1 liter of water,adjusting the pH to 7.0 and autoclaving). The antibiotic kanamycin (30mg) was added aseptically after autoclaving. A portion of the resultingculture (0.2 ml) was then transferred to Terrific Broth (TB, 10 ml,prepared by dissolving bacto-tryptone (12 g), bacto-yeast extract (12g), and glycerol (4 ml) in deionized water (900 ml), autoclaving andthen adding a sterile solution (100 ml) containing 50 μg/ml ampicillin,0.17 M KH₂PO₄, and 0.72 M K₂HP0₄) and allowed to grow until the opticaldensity at 260 nm (OD²⁶⁰) reached 0.6. This culture was used toinoculate 50 ml of TB containing 50 μg/ml of ampicillin, which was thenplaced at 28° C. and 200 rpm and a heme precursor, δ- aminolevulimicacid (1 mM), was added followed by the inducer IPTG (0.4 mM) one hourlater. The induced cultures were left for a further period of time (4 or16 hours) and the cells harvested by centrifugation (5,000 rpm for 7 minat 4° C.). The precipitated cells were washed by resuspending them inTris-HC1 buffer (50 mM, pH 7.9) followed by recentrifugation as before.

The resulting pellet of cells was resuspended in Tris-acetate buffer(0.1 M, pH 7.6) containing sucrose (0.5 M), EDTA (0.5 mM) and lysozyme(1 mg/ml). After 30 min on ice, the mixture was centrifuged as before toobtain a pellet of spheroplastes. These were resuspended in potassiumphosphate buffer (0.1 M, pH 7.6) containing magnesium acetate (6 mM),glycerol (20% v/v) and DTT (0.1 mM) and the mixture left for 10 min at−80° C. Following this, a protease inhibitor was added (PMSF, 1 mM) andthe cells sonicated (2×30 seconds). Analysis of the expression productsby SDS-PAGE showed barely detectable bands for both Clones B and C.Compared to the control protein produced from vector alone with no cDNAinsert, there was less protein, but the bacterial lysates of each gaveeasily measurable catalytic activity. By monitoring the disappearance ofthe UV-235nm absorbance of the fatty acid hydroperoxide substrates, lessthan 1 μl (<10 μg crude protein) of the suspended and lysed bacterialpellets were required in order to observe reaction in a 1 ml UV cuvette.

Example 4

Partial purification of the 9-HPL derived from clone C

The 9-HPL enzyme was expressed in E. coli (BL21 cells), as discussed inExample 3, however, a His-6 tag was expressed on the carboxyl terminusof the protein using the nucleotide sequence of SEQ ID NO:31. Thepreparations of solubilized spheroplastes from three 50 ml bacterialcultures were pooled and applied to a nickel-NTA column (purchased fromQiagen) according to the manufacturer's instructions. The column (bedvolume 1 ml) was washed with the application buffer (containing 50 mMglycine and 0.1% Emulphogen) and the enzyme was then eluted using theapplication buffer containing 40 mM histidine and 0.1% Emulphogendetergent. The pooled fractions wcre subsequently dialyzed overnight toremove the histidine. This gave approximately 5 ml of solution, which byanalysis on SDS-PAGE, contained the expected 55 kD band of the 9-HPL asthe main protein component. The UV-visible spectrum of the partiallypurified 9-HPL showed a main Soret band of the hemoprotein with anabsorbance of 0.35 AU at 416 nm.

Example 5

Catalytic activities of the expressed melon Clone C

A. Turnover number of the 9-HPL using 9S-hydroperoxylinoleic acid, atroom temperature, pH 7.6 Measurement was made using thespectrophotometric assay (decrease in absorbance at 235 nm) and theinitial rates of reaction. The turnover number of the purified 9-HPLenzyme (number of product molecules formed per molecule of enzyme) using9S-hydroperoxylinoleic acid as substrate was calculated from the knownconcentration of the enzyme (measured at the Soret maximum at 416 nm,and using a molar extinction coefficient of 100,000), and the measuredrates of change of substrate concentration (using the molar extinctioncoefficient of 23,000 at 235 nm of the conjugated diene). The valuesobtained were 3000 turnovers per second for the most active preparationof the 9-HPL enzyme.

This calculation refers to the observed initial rates of reaction. Therates decreased with time as the enzyme undergoes a turnover-dependentinactivation.

B. Identification of products formed by the purified 9-HPL enzyme from9S-hydroperoxylinoleic acid The purified enzyme (approximately 0.4 μg in2 μl) was reacted with 3 μg [U-14C]9S-hydroperoxylinoleic acid in ¹⁰⁰ μlof buffer (potassium phosphate, 0.1 M, pH 7.6). After 30 seconds at roomtemperature, at which time reaction was complete, methanol (200 μl) wasadded. The solution was mixed, briefly spun in a bench-top centrifuge,and the supernatant injected on HPLC.

The HPLC system used a Beckman Ultrasphere 5 μm ODS column (25×0.46 cm),a solvent of methanol/watcr/glacial acetic acid (75/25/0.01, v/v/v), anda flow rate of 1.1 ml/min. The column was coupled to a Hewlett-Packard1040A diode array detector for detection of UV absorbing compounds, andthen the eluant was passed through a Packard Flo-One radioactive on-linedetector for recording the profile of ¹⁴C metabolites.

The substrate, uniformly labeled with ¹⁴C, was converted to two mainradiolabeled products, which were equal in area. The early elutingproduct (at 3.5 min retention time was identified subsequently by GC-MSas 9-oxo-nonanic acid (see below); this product represents the first 9carbons of the 18 carbon substrate. The second main product, at aretention time of 9 min, coincided precisely in retention time with3Z-nonenal. This product represents carbons 10-18 of the substrate. Avery small back shoulder on this peak, approximately 5% of the peakarea, coincided with authentic 2E-nonenal.

C. Identification of 9-oxo-nonanic acid

The early eluting product (3.5 min retention time) from reaction of the9-HPL with 9S-hydroperoxylinoleic acid exhibited only weak endabsorbance in the UV. This product was purified using the HPLC systemdescribed above and was extracted from the column solvent with diethylether. An aliquot was redissolved in 20 μl of methanol and treated withethereal diazomethane to convert the free acid to the methyl ester. Partof this methylated sample was also converted to the methoxime derivativeby treatment of the sample with 2% methoxylamine hydrochloride (MOX) inpyridine.

The two samples (methyl ester and methyl ester-methoxime derivatives)were analyzed by GC-MS (gas chromatography-mass spectrometry) operatedin the electron impact mode using a Finnigan Ineos 50 mass spectrometercoupled to a Hewlett-Packard 5890 gas chromatograph equipped with aSPB-5 fused silica capillary column (30 m×0.25 mm internal diameter).Samples were injected at 50° C. and the temperature was subsequentlyprogrammed to 300° C. at 10°/min. Under these conditions, 9-oxo-nonanicacid methyl ester eluted at 13 minutes retention time. The mass spectrumshowed characteristic fragments at m/z 185 (M+—H), 158 (M+—CO), 155(M+—OCH₃), 143 (M+—CH₂CHO), 111 and the methyl ester McLafferty fragmentions at m/z 74 and 87. MOX-derivatization of the methyl ester yielded adouble gas chromatographic peak comprised of the syn- and anti- oximeisomers which eluted together at about 14.5 minutes. Their mass spectrashowed the same main fragment ions with slight differences in ionintensities. Major ions were detected at m/z 215 (M+), 184 (M+—NH₂OCH₃),152 (M+—NH₂OCH₃—CH₃OH) 124 (184 —CH₃CO₂H) and 73 (CH₃—CNH—OCH₃+).

D. Identification of 3Z-nonenal

A reaction of 9S-hydropcroxylinolcic acid with the purified 9-HPL wasextracted with hexane and an aliquot of the hexane extract was injectedon the GC-MS system described above. Two peaks eluted on GC-MS at theretention times of authentic standards of 3Z-nonenal (≈8 minutes) and2E-nonenal (≈9 minutes). As judged by peak area, the two aldehydes wereformed in a ratio of 10:1 of 3Z to 2E. For identification of the twoaldehydes, a standard of 3Z-nonenal was chemically synthesized (seeExample 6), and 2E-nonenal was purchased from Aldrich (Milwakee, Wis.).The mass spectra for both aldehydes produced by the 9-HPL reaction with9S-hydroperoxylinoleic acid are virtually identical with the authenticstandards. 3Z-Nonenal shows characteristic fragment ions at m/z 140(M+), 122 (M+—H₂O) and 111 (M+—CHO)), while 2E-nonenal showed ions atm/z 139 (M+—H), 122 (M+—H₂O) and 111 (M+—CHO).

Example 6

Chemical synthesis of 3Z-nonenal

3Z-nonenal synthesis was carried out by slight modifications of themethods of Corey and Suggs (1975), and Andre and Funk (1986). Briefly,to a NaOAc-buffered solution of pyridiniumchlorochromate in methylenechloride, 3Z-nonenol dissolved in methylene chloride was added. Afterstirring at room temperature, the reaction was terminated by addition ofdiethyl ether and immediately filtered through a column of silica geleluted with methylene chloride to remove the oxidizing agent. TLCanalysis indicated that conversion to 3Z-nonenal was about 50% complete.The crude product was isolated by open bed column chromatography andpurified by RP-HPLC. At all steps during purification, care was taken toprevent oxidation of 3Z-nonenal to 4-hydroperoxy-2E-nonenal. A GC-MSanalysis of the chemically synthesized 3Z-nonenal showed that the massspectrum of the chemically synthesized 3Z-nonenal is virtually identicalwith the authentic standard, showing the characteristic fragment ions.

Example 7

Identification of products formed by the 9-HPL enzyme in the crudebacterial lysate from 9S-hydroperoxylinoleic acid

When the crude lysate of the bacterial expression was used as a sourceof 9-HPL we obtained a different product profile compared to thatobtained using the purified enzyme. The analytical studies describedbelow (particularly the trapping experiment) led to the conclusion thatthe initial enzymatic products were identical to those characterizedusing the purified enzyme. However, in the crude bacterial lysatc, oneof the two primary enzymatic products, 3Z-nonenal, is readily oxidized(probably non-enzymatically) to a mixture of three aldehydes comprisedof 4-hydroxy-2E-nonenal (4-HNE), 4-hydroperoxy-2E-nonenal (4-HPNE), anda hemiacetal derivative formed between 9-oxo-nonanic acid and4-hydroperoxy-2E-nonenal (hemiacetal). The structures of the three polaraldehydes and their formation from 3Z-nonenal are depicted in FIG. 6.This also shows the minor isomerization of 3Z-nonenal to 2E-nonenalwhich is observed to a small extent using either the purified enzyme orthe crude bacterial lysate. In the crude bacterial lysate, the otherprimary 9-HPL product, 9-oxo-nonanic acid, is recovered mainlyunchanged. A small fraction is converted to the hemiacetal as depictedin FIG. 6.

Using the crude bacterial lysate expressing the melon 9-HPL, reactionswith 9S-hydroperoxylinoleic acid were monitored using an oxygenelectrode (the electrode records O₂ concentration in solution versustime). It was observed by conducting incubations in the closed 2 ml cellof the oxygen electrode that reactions of the 9-HPL from the crudelysate with 9S-hydroperoxylinoleic acid were associated with a fall inO₂ concentration in the solution. This reduction in the O₂ concentrationcorresponds to the reaction of O₂ with 3Z-noncnal to give the threepolar aldehydes. Quantitatively, the fall in O₂ concentration (nmole O₂consumed) corresponded approximately to the nmole of polar aldehydederivatives detected by HPLC analysis. By contrast to the crude enzymepreparation, reactions of the purified 9-HPL with 9S-hydroperoxylinoleicacid were associated with no change in O₂ concentration in solution.

Using the crude bacterial lysate expressing the melon 9-HPL, reactionswith 9S-hydroperoxylinoleic acid were monitored either using the O₂electrode or spectrophotometrically at 235 nm as described above. Thesolutions were then extracted using a C18 extraction cartridge(Bond-Elut from Varian), and eluted using diethyl ether. The etherextracts were evaporated to dryness and analyzed by HPLC. The profile ofradiolabeled products was obtained using [1-¹⁴C]9S-hydroperoxylinoleicacid (¹⁴C on carbon-1) and [U-¹⁴C]9S-hydroperoxylinoleic acid (¹⁴Cuniformly on all 18 carbons) as substrate. The profile of UV-absorbingmaterials was detected by monitoring at 205 nm and 220 nm. When using1-¹⁴C substrate, only products retaining carbon-1 of the substrate areradiolabeled (i.e. 9-oxo-nonanic acid and the hemiacetal product), andfrom U-¹⁴C substrate, all products are radiolabeled.

The largest radiolabeled peak, formed from both the 1-¹⁴C and theuniformly-labeled ¹⁴C substrate, was identified as 9-oxo-nonanic acid.This corresponds to carbons 1-9 of the original substrate and thisprimary aldehydic product of the 9-HPL is recovered mainly intact fromthe incubations. A small amount is converted to hemiacetal as shown inFIG. 6.

The three products are derived via the initial oxygenation of3Z-nonenal. This oxidation of 3Z-nonenal, initially to form4-hydroperoxy-2E-nonenal (4-HPNE), is probably a non-enzymatic reactionthat occurs readily in the crude bacterial lysate. The 4-HPNE is partlyreduced to 4-HNE. The 4-HPNE also reacts with 9-oxo-nonanic acid to formthe hemiacetal derivative (FIG. 6).

Example 8

Evidence that the primary products of the 9-HPL in the crude bacteriallysate are 9-oxo-nonanic acid and 3Z-nonenal

For this series of experiments, prior to reaction with the crude 9-HPL,the oxygen concentration in the buffer was reduced to zero. This wasaccomplished by addition of small aliquots of a solution of sodiumdithionite while monitoring the 02 concentration using the oxygenelectrode.

Using buffer depleted of oxygen, it was shown that the rate of reactionof the 9-HPL with 9S-hydroperoxylinoleic acid was not decreased by theabsence of O₂. This was demonstrated using the spectrophotomctric assay(rate of disappearance of the UV absorbance at 235 nm).

Reaction of [U-¹⁴C]9S-hydropcroxylinoleic acid (40 μg) with 9-HPL fromthe crude bacterial lysate was carried out in O₂-depleted buffer in the2 ml cell of the oxygen electrode. After 1 minute, at which time thereaction was expected to be almost complete, 50 μl of a freshly prepared10 mg/ml solution of NaBH₄ was injected and the reduction reactionallowed to proceed for 5 minutes. This procedure immediately reduced(and thereby stabilized) the aldehydes as the corresponding alcohols(9-hydroxy-nonanic acid and 3Z-nonenol).

The 2ml solution was subsequently extracted using a C18 extractioncartridge (Bond-Elut, from Varian) and the products recovered by elutionwith diethyl ether. 50 μg of unlabeled authentic 3Z-nonenol and 50 μg2E-nonenol (obtained from Aldrich) were added to an aliquot of thesample and the sample was then analyzed by HPLC.

One chromatogram showed the radiolabeled products and anotherchromatogram depicted the UV profile at 205 nm. The two main peaks inthe UV chromatogram corresponded to the two added standards and thusestablish the precise retention times of 3Z-nonenol and 2E-nonenol. Thelater peaks in the UV chromatogram correspond to the reduction productof unused substrate (9-hydroxy-linoleic acid) and its 10 trans-12transisomer that may have been a minor contaminant of the original substrate.

The ¹⁴C chromatogram showed an early eluting peak at 3 minutesidentified as 9-hydroxy-nonanic acid, the NaBH₄-reduction product of theprimary enzymatic product, 9-oxo-nonanic acid. The second mainradiolabeled peak, eluting at 8.8 minutes, corresponded to 3Z-nonenol,the NaBH₄-reduction product of 3Z-nonenal. 2E-Nonenol was not detectedin the NaBH₄-trapping experiment. This suggested that the correspondingaldehyde, 2E-nonenal, was not a primary enzymatic product, but ratherwas formed by non-enzymatic isomerization. In the NaBH₄-trappingexperiment, its formation was reduced due to the prompt conversion ofthe 3Z-nonenal to the more stable alcohol.

The results of the trapping experiment indicate that the activity of the9-HPL in the crude bacterial lysatc was restricted to conversion of9S-hydroperoxy-linoleic acid to the two primary aldehydes, 9-oxo-nonanicacid and 3Z-nonenal. The other aldehydes recovered from reactions of the9-HPL in the crude bacterial lysate were formed by subsequent reactionsof the primary products with molecular oxygen or by isomerization to2E-nonenal.

Example 9

Identification of 4-hydroperoxy-2E-nonenal (4-HPNE) and4-hydroxy-2E-nonenal (4-HNE)

From the incubations described in Example 7, 4-HPNE was isolated byreversed-phase HPLC and characterized by ¹H-NMR spectroscopy (9.58 ppm,d, J=7.8, H1; 6.9 ppm, dd, J=15.9, 6.2, H3; 6.25, ddd, J=15.9, 7.8, 1.2,H2; 4.6 ppm, q (with some fine structure), J>>6.5, H4). Formation of4-hydroxy-2E-nonenal (4-HNE) was also seen in the bacterial lysatereactions where it was formed by non-specific reduction of 4-HPNE (seeExample 7). The 4-HNE recovered from enzyme incubations was identical inits UV spectrum and HPLC retention times to an authentic sample of 4-HNEobtained from Cayman Chemical Co. (Ann Arbor, Mich.).

For mass spectrometric characterization of 4-HPNE, an aliquot wasreduced using triphenyphosphine to the corresponding alcohol, 4-HNE andrepurified by HPLC. Using the previously described GC-MS system, the4-HNE was analyzed directly and after treatment with BSTFA to give thetrimethylsilyl ether derivative. The fragment ions obtained for thenon-derivatized 4-HNE are in accord with reports in the literature(Gardner et al., 1992). Specifically, the following fragment ions wereobserved: m/z 138 (M+—H₂O), 127 (M+—CHO), 109 (M+—CHO —H₂O), 99, 86, and85. The trimethylsilyl ether derivative showed diagnostic ions at m/z199 (M+—CHO), 157 (CHO—C₂H₂—CH—OSi(CH₃)3+) and 129(CHO—C₂H₂—CH—OSi(CH₃)3+—CO).

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

References

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SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 56 <210> SEQ ID NO 1 <211>LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Cucumis melo <400> SEQUENCE:1 Met Ala Thr Pro Ser Ser Ser Ser Pro Glu 1 5 10 <210> SEQ ID NO 2 <211>LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Cucumis melo <400> SEQUENCE:2 Ile Leu Phe Asp Thr Ala Lys Val Glu Lys Arg Asn Ile Leu Asp 1 5 10 15<210> SEQ ID NO 3 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:Cucumis melo <400> SEQUENCE: 3 Arg Leu Phe Leu Ser Phe Leu Ala 1 5 <210>SEQ ID NO 4 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Cucumis melo<400> SEQUENCE: 4 Ser Ile Ser Asp Ser Met Ser 1 5 <210> SEQ ID NO 5<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Cucumis melo <400>SEQUENCE: 5 Leu Leu Ser Asp Gly Thr Pro Asp 1 5 <210> SEQ ID NO 6 <211>LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Cucumis melo <400> SEQUENCE:6 Ile Phe Ser Val Phe Glu Asp Leu Val Ile 1 5 10 <210> SEQ ID NO 7 <211>LENGTH: 481 <212> TYPE: PRT <213> ORGANISM: Cucumis melo <400> SEQUENCE:7 Met Ala Thr Pro Ser Ser Ser Ser Pro Glu Leu Pro Leu Lys Pro Ile 1 5 1015 Pro Gly Gly Tyr Gly Phe Pro Phe Leu Gly Pro Ile Lys Asp Arg Tyr 20 2530 Asp Tyr Phe Tyr Phe Gln Gly Arg Asp Glu Phe Phe Arg Ser Arg Ile 35 4045 Thr Lys Tyr Asn Ser Thr Val Phe Arg Ala Asn Met Pro Pro Gly Pro 50 5560 Phe Ile Ser Ser Asp Ser Arg Val Val Val Leu Leu Asp Ala Leu Ser 65 7075 80 Phe Pro Ile Leu Phe Asp Thr Ala Lys Val Glu Lys Arg Asn Ile Leu 8590 95 Asp Gly Thr Tyr Met Pro Ser Leu Ser Phe Thr Gly Asn Ile Arg Thr100 105 110 Cys Ala Tyr Leu Asp Pro Ser Glu Thr Glu His Ser Val Leu LysArg 115 120 125 Leu Phe Leu Ser Phe Leu Ala Ser Arg His Asp Arg Phe IlePro Leu 130 135 140 Phe Arg Ser Ser Leu Ser Glu Met Phe Val Lys Leu GluAsp Lys Leu 145 150 155 160 Ser Glu Lys Lys Lys Ile Ala Asp Phe Asn SerIle Ser Asp Ser Met 165 170 175 Ser Phe Asp Tyr Val Phe Arg Leu Leu SerAsp Gly Thr Pro Asp Ser 180 185 190 Lys Leu Ala Ala Glu Gly Pro Gly MetPhe Asp Leu Trp Leu Val Phe 195 200 205 Gln Leu Ala Pro Leu Ala Ser IleGly Leu Pro Lys Ile Phe Ser Val 210 215 220 Phe Glu Asp Leu Val Ile HisThr Ile Pro Leu Pro Phe Phe Pro Val 225 230 235 240 Lys Ser Gly Tyr ArgLys Leu Tyr Glu Ala Phe Tyr Ser Ser Ser Gly 245 250 255 Ser Phe Leu AspGlu Ala Glu Lys Gln Gly Ile Asp Arg Glu Lys Ala 260 265 270 Cys His AsnLeu Val Phe Leu Ala Gly Phe Asn Ala Tyr Gly Gly Met 275 280 285 Lys ValLeu Phe Pro Thr Leu Leu Lys Trp Val Gly Thr Ala Gly Glu 290 295 300 AspLeu His Arg Lys Leu Ala Glu Glu Val Arg Thr Thr Val Lys Glu 305 310 315320 Glu Gly Gly Leu Thr Phe Ser Ala Leu Glu Lys Met Ser Leu Leu Lys 325330 335 Ser Val Val Tyr Glu Ala Leu Arg Ile Glu Pro Pro Val Pro Phe Gln340 345 350 Tyr Gly Lys Ala Lys Glu Asp Ile Val Ile Gln Ser His Asp SerSer 355 360 365 Phe Lys Ile Lys Lys Gly Glu Thr Ile Phe Gly Tyr Gln ProPhe Ala 370 375 380 Thr Lys Asp Pro Lys Ile Phe Lys Asp Ser Glu Lys PheVal Gly Asp 385 390 395 400 Arg Phe Val Gly Glu Glu Gly Glu Lys Leu LeuLys Tyr Val Tyr Trp 405 410 415 Ser Asn Glu Arg Glu Thr Val Glu Pro ThrAla Glu Asn Lys Gln Cys 420 425 430 Pro Gly Lys Asn Leu Val Val Leu IleGly Arg Ile Met Val Val Glu 435 440 445 Phe Phe Leu Arg Tyr Asp Thr PheThr Val Glu Val Ala Asp Leu Pro 450 455 460 Leu Gly Pro Ala Val Lys PheLys Ser Leu Thr Arg Ala Thr Asp Met 465 470 475 480 Val <210> SEQ ID NO8 <211> LENGTH: 1446 <212> TYPE: DNA <213> ORGANISM: Cucumis melo <400>SEQUENCE: 8 atggctactc cttcttcctc ctcccctgaa cttcctctca aaccaattcccggtggctat 60 ggcttcccct tcctcggtcc catcaaagac cgttacgatt acttctatttccaaggtaga 120 gacgaattct tccgttcccg gattaccaaa tacaactcca ccgtcttccgcgccaacatg 180 ccaccgggcc ccttcatttc ctccgattcc agagtcgttg tccttctcgatgccctcagt 240 tttcctatcc tcttcgacac agccaaagtc gagaaacgca acattctcgacggaacttac 300 atgccctcct tgtccttcac cggcaacatt cgcacctgtg cttatttggacccatcggaa 360 acagagcact ctgttctcaa acgcctcttc ctctcctttc tcgcttcccgccatgacagg 420 ttcatccctc tgtttcgaag ctccttgtct gagatgtttg ttaagcttgaagataaactt 480 tccgagaaaa agaagatcgc tgatttcaac tcgatcagcg attccatgtcgtttgattat 540 gttttccgtt tactctccga tggaacccct gattcgaaat tagctgctgagggacctgga 600 atgttcgatc tgtggcttgt gtttcaactc gccccattgg cttccattggccttcccaaa 660 attttctctg tttttgaaga tctcgtcatt cacaccattc ccctgcctttcttcccagtc 720 aagagtggtt acaggaagct ttatgaagcg ttttactcct cttctggctcatttctagac 780 gaagcagaga aacaggggat agacagggag aaagcatgtc acaatttagtgtttctcgct 840 ggattcaacg catacggggg aatgaaagtc ctttttccca ctttactgaaatgggtcggc 900 accgccggcg aggatctcca ccggaaactc gccgaggaag tcaggacaaccgtgaaggaa 960 gaagggggac tgactttctc cgccttggag aaaatgagtc tgctgaagtccgtcgtgtac 1020 gaagcactca ggatcgaacc gccggtgccg ttccagtacg ggaaagcgaaggaggatatc 1080 gtgattcaga gccacgattc ttctttcaag atcaaaaaag gggagacgatttttggttat 1140 cagccgtttg ctactaaaga tccgaagatt tttaaggatt cggagaagttcgtgggcgat 1200 aggttcgtgg gagaggaagg ggagaagctt ttgaagtatg tttactggtcaaatgagcgg 1260 gagacagtgg agccgacggc ggagaacaag cagtgtccgg ggaagaatctggtggtgctg 1320 ataggtagga ttatggtggt ggaattcttc cttcgttatg atacgttcaccgtggaggtc 1380 gcagatttgc cgctgggtcc ggcagtgaag ttcaagtcct taaccagagcaaccgatatg 1440 gtttaa 1446 <210> SEQ ID NO 9 <211> LENGTH: 60 <212>TYPE: PRT <213> ORGANISM: Psidium Guava <400> SEQUENCE: 9 Gly Glu LeuLeu Cys Gly Tyr Gln Lys Val Val Met Thr Asp Pro Lys 1 5 10 15 Val PheAsp Glu Pro Glu Ser Phe Asn Ser Asp Arg Phe Val Gln Asn 20 25 30 Ser GluLeu Leu Asp Tyr Leu Tyr Trp Ser Asn Gly Pro Gln Thr Gly 35 40 45 Thr ProThr Glu Ser Asn Lys Gln Cys Ala Ala Lys 50 55 60 <210> SEQ ID NO 10<211> LENGTH: 61 <212> TYPE: PRT <213> ORGANISM: Banana <400> SEQUENCE:10 Gly Glu Leu Leu Cys Gly Tyr Gln Pro Leu Val Met Arg Asp Pro Ala 1 510 15 Val Phe Asp Asp Pro Glu Thr Phe Ala Pro Glu Arg Phe Met Gly Ser 2025 30 Gly Lys Glu Leu Leu Lys Tyr Val Phe Trp Ser Asn Gly Pro Glu Thr 3540 45 Gly Thr Pro Thr Pro Ala Asn Lys Gln Cys Ala Ala Lys 50 55 60 <210>SEQ ID NO 11 <211> LENGTH: 62 <212> TYPE: PRT <213> ORGANISM: Capsicumannum (green pepper) <400> SEQUENCE: 11 Gly Glu Leu Leu Cys Gly Tyr GlnPro Leu Val Met Lys Asp Pro Lys 1 5 10 15 Val Phe Asp Glu Pro Glu LysPhe Met Leu Glu Arg Phe Thr Lys Glu 20 25 30 Lys Gly Lys Glu Leu Leu AsnTyr Leu Phe Trp Ser Asn Gly Pro Gln 35 40 45 Thr Gly Ser Pro Thr Glu SerAsn Lys Gln Cys Ala Ala Lys 50 55 60 <210> SEQ ID NO 12 <211> LENGTH: 62<212> TYPE: PRT <213> ORGANISM: Arabidopsis <400> SEQUENCE: 12 Gly GluMet Leu Tyr Gly Tyr Gln Pro Leu Ala Thr Arg Asp Pro Lys 1 5 10 15 IlePhe Asp Arg Ala Asp Glu Phe Val Pro Glu Arg Phe Val Gly Glu 20 25 30 GluGly Glu Lys Leu Leu Arg His Val Leu Trp Ser Asn Gly Pro Glu 35 40 45 ThrGlu Thr Pro Thr Val Gly Asn Lys Gln Cys Ala Gly Lys 50 55 60 <210> SEQID NO 13 <211> LENGTH: 61 <212> TYPE: PRT <213> ORGANISM: Flax <400>SEQUENCE: 13 Gly Glu Met Leu Phe Gly Tyr Gln Pro Phe Ala Thr Lys Asp ProLys 1 5 10 15 Ile Phe Asp Arg Pro Glu Glu Phe Val Ala Asp Arg Phe ValGly Glu 20 25 30 Gly Val Lys Leu Met Glu Tyr Val Met Trp Ser Asn Gly ProGlu Thr 35 40 45 Glu Thr Pro Ser Val Ala Asn Lys Gln Cys Ala Gly Lys 5055 60 <210> SEQ ID NO 14 <211> LENGTH: 61 <212> TYPE: PRT <213>ORGANISM: Guayule <400> SEQUENCE: 14 Gly Glu Met Leu Phe Gly Tyr Gln ProPhe Ala Thr Lys Asp Pro Lys 1 5 10 15 Val Phe Asp Arg Pro Glu Glu PheVal Ala Asp Arg Phe Val Gly Glu 20 25 30 Gly Val Lys Leu Met Glu Tyr ValTrp Trp Ser Asn Gly Pro Glu Thr 35 40 45 Glu Ser Pro Thr Val Glu Asn LysGln Cys Ala Gly Lys 50 55 60 <210> SEQ ID NO 15 <211> LENGTH: 487 <212>TYPE: PRT <213> ORGANISM: Cucumis melo <221> NAME/KEY: VARIANT <222>LOCATION: (1)...(487) <223> OTHER INFORMATION: Xaa = Any Amino Acid<221> NAME/KEY: misc_feature <222> LOCATION: (0)...(0) <223> OTHERINFORMATION: Accession No. AF081955 <400> SEQUENCE: 15 Met Ala Thr ProSer Ser Ser Ser Pro Glu Leu Pro Leu Lys Pro Ile 1 5 10 15 Pro Gly GlyTyr Gly Phe Pro Phe Leu Gly Pro Ile Lys Asp Arg Tyr 20 25 30 Asp Tyr PheTyr Phe Gln Gly Arg Asp Glu Phe Phe Glu Arg Ser Arg 35 40 45 Ile Thr LysTyr Asn Ser Thr Val Phe Arg Ala Asn Met Pro Pro Gly 50 55 60 Pro Phe IleSer Ser Asp Ser Arg Val Val Val Leu Leu Asp Ala Leu 65 70 75 80 Ser PhePro Ile Leu Phe Asp Thr Ala Lys Val Glu Lys Arg Asn Ile 85 90 95 Leu AspGly Thr Tyr Met Pro Ser Leu Ser Phe Thr Gly Asn Ile Arg 100 105 110 ThrCys Ala Tyr Leu Asp Pro Ser Glu Thr Glu His Ser Val Leu Lys 115 120 125Arg Leu Phe Leu Ser Phe Leu Ala Ser Arg His Asp Arg Phe Ile Pro 130 135140 Leu Phe Arg Ser Ser Leu Ser Glu Met Phe Val Lys Leu Glu Asp Lys 145150 155 160 Leu Ser Glu Lys Lys Lys Ile Ala Asp Phe Asn Ser Ile Ser AspSer 165 170 175 Met Ser Phe Asp Tyr Val Phe Arg Leu Leu Ser Asp Gly ThrPro Asp 180 185 190 Ser Lys Leu Ala Ala Glu Gly Pro Gly Met Phe Asp LeuTrp Leu Val 195 200 205 Phe Gln Leu Ala Pro Leu Ala Ser Ile Gly Leu ProLys Ile Phe Ser 210 215 220 Val Phe Glu Asp Leu Val Ile His Thr Ile ProLeu Pro Phe Phe Pro 225 230 235 240 Val Lys Ser Gly Tyr Arg Lys Leu TyrGlu Ala Phe Tyr Ser Ser Ser 245 250 255 Gly Ser Phe Leu Asp Glu Ala GluLys Gln Gly Ile Asp Arg Glu Lys 260 265 270 Ala Cys His Asn Leu Val PheLeu Ala Gly Phe Asn Ala Tyr Gly Gly 275 280 285 Met Lys Val Leu Phe ProThr Leu Leu Lys Trp Val Gly Thr Ala Gly 290 295 300 Glu Asp Leu His ArgLys Leu Ala Glu Glu Val Arg Thr Thr Val Lys 305 310 315 320 Glu Glu GlyGly Leu Thr Phe Ser Ala Leu Glu Lys Met Ser Leu Leu 325 330 335 Lys SerVal Val Tyr Glu Ala Leu Arg Ile Glu Pro Pro Val Pro Phe 340 345 350 GlnTyr Gly Lys Ala Lys Glu Asp Ile Val Ile Gln Ser His Asp Ser 355 360 365Ser Phe Lys Ile Lys Lys Gly Glu Thr Ile Phe Gly Tyr Gln Pro Phe 370 375380 Ala Thr Lys Asp Pro Lys Ile Phe Lys Asp Ser Glu Lys Phe Val Gly 385390 395 400 Asp Arg Phe Val Gly Glu Glu Gly Glu Lys Leu Leu Lys Tyr ValTyr 405 410 415 Trp Ser Asn Glu Arg Glu Thr Val Glu Pro Thr Arg Xaa AsnLys Gln 420 425 430 Cys Pro Gly Lys Asn Leu Val Val Leu Ile Gly Arg IleMet Val Val 435 440 445 Glu Phe Phe Leu Arg Tyr Asp Thr Phe Thr Val GluVal Ala Asp Leu 450 455 460 Pro Leu Gly Pro Ala Val Lys Phe Lys Ser LeuThr Arg Ala Thr Asp 465 470 475 480 Met Leu Lys Leu Met Thr Asn 485<210> SEQ ID NO 16 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (1)...(23) <223> OTHER INFORMATION: n = A,T,C or G y = C orT(U) <223> OTHER INFORMATION: Description of Artificial Sequence:/Note =synthetic construct <400> SEQUENCE: 16 ggtgagttgc tntgyggnta yca 23<210> SEQ ID NO 17 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (1)...(20) <223> OTHER INFORMATION: n = A,T,C or G <223> OTHERINFORMATION: y = T,C <223> OTHER INFORMATION: Description of ArtificialSequence:/Note = synthetic construct <400> SEQUENCE: 17 ggtgagttgctntgyggnta 20 <210> SEQ ID NO 18 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (1)...(20) <223> OTHER INFORMATION: n =A,T,C or G y = C or T(U) <223> OTHER INFORMATION: Description ofArtificial Sequence:/Note = synthetic construct <400> SEQUENCE: 18tggtcnaayg gnccrgagac 20 <210> SEQ ID NO 19 <211> LENGTH: 23 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (1)...(23) <223> OTHER INFORMATION: n =A,T,C or G y = C or T(U) r = A or G <223> OTHER INFORMATION: Descriptionof Artificial Sequence:/Note = synthetic construct <400> SEQUENCE: 19tactggtcna ayggnccnsa rac 23 <210> SEQ ID NO 20 <211> LENGTH: 24 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (1)...(24) <223> OTHERINFORMATION: n = A,T,C or G y = C or T(U) r = A or G <223> OTHERINFORMATION: Description of Artificial Sequence:/Note = syntheticconstruct <400> SEQUENCE: 20 aayaarcart gygcngctaa ggac 24 <210> SEQ IDNO 21 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(1)...(21) <223> OTHER INFORMATION: n = A,T,C or G y = C or T(U) r = Aor G <223> OTHER INFORMATION: Description of Artificial Sequence:/Note =synthetic construct <400> SEQUENCE: 21 aarcartgyg cngctaagga c 21 <210>SEQ ID NO 22 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence:/Note = synthetic construct <400> SEQUENCE: 22 GlyGlu Leu Leu Cys Gly Tyr Gln 1 5 <210> SEQ ID NO 23 <211> LENGTH: 7 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence:/Note = syntheticconstruct <400> SEQUENCE: 23 Gly Glu Leu Leu Cys Gly Tyr 1 5 <210> SEQID NO 24 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence:/Note = synthetic construct <400> SEQUENCE: 24 TrpSer Asn Gly Pro Glu Thr 1 5 <210> SEQ ID NO 25 <211> LENGTH: 8 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence:/Note = syntheticconstruct <400> SEQUENCE: 25 Tyr Trp Ser Asn Gly Pro Glu Thr 1 5 <210>SEQ ID NO 26 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION:(1)...(8) <223> OTHER INFORMATION: Xaa = Any Amino Acid <223> OTHERINFORMATION: Description of Artificial Sequence:/Note = syntheticconstruct <400> SEQUENCE: 26 Asn Lys Gln Cys Ala Ala Xaa Xaa 1 5 <210>SEQ ID NO 27 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION:(1)...(7) <223> OTHER INFORMATION: Xaa = Any Amino Acid <223> OTHERINFORMATION: Description of Artificial Sequence:/Note = syntheticconstruct <400> SEQUENCE: 27 Lys Gln Cys Ala Ala Xaa Xaa 1 5 <210> SEQID NO 28 <211> LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Cucumis melo<400> SEQUENCE: 28 Gly Glu Leu Leu Cys Gly Tyr Gln Pro Leu Val Met ArgAsp Pro Lys 1 5 10 15 Val Phe Asp Glu Pro Glu Ala Phe Asn Pro Asp ArgPhe Arg Gly Glu 20 25 30 <210> SEQ ID NO 29 <211> LENGTH: 32 <212> TYPE:PRT <213> ORGANISM: Cucumis melo <400> SEQUENCE: 29 Gly Glu Leu Leu CysGly Tyr Gln Pro Phe Ala Thr Arg Asp Pro Lys 1 5 10 15 Ile Phe Asp ArgAla Asp Glu Phe Val Pro Asp Arg Phe Thr Gly Glu 20 25 30 <210> SEQ ID NO30 <211> LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Cucumis melo <400>SEQUENCE: 30 Gly Glu Leu Leu Cys Gly Tyr Gln Pro Phe Ala Thr Lys Asp ProLys 1 5 10 15 Ile Phe Lys Asp Ser Glu Lys Phe Val Gly Asp Arg Phe ValGly Glu 20 25 30 <210> SEQ ID NO 31 <211> LENGTH: 272 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence:/Note = syntheticconstruct <400> SEQUENCE: 31 agctaatgac taattagttt tatcatttac agatagtgaattggttgatg cacggaagct 60 gtggcggact gcgcacacat gattgagtac ttggggttattaaagtaatt tcgttgtgat 120 ccacgtggtc ttattttaat ttgagatctc attgtgtgttgtaacccacc ggtcatctta 180 ttttatagtt tgtttgtttt ctcaattatg ctccaaattttaaaataaat aaataccatc 240 ttcttctttt tactaaaaaa aaaaaaaaaa aa 272 <210>SEQ ID NO 32 <211> LENGTH: 480 <212> TYPE: PRT <213> ORGANISM: Capsicumannum (green pepper) <400> SEQUENCE: 32 Met Ile Pro Ile Met Ser Ser AlaPro Leu Ser Thr Ala Thr Pro Ile 1 5 10 15 Ser Leu Pro Val Arg Lys IlePro Gly Ser Tyr Gly Phe Pro Leu Leu 20 25 30 Gly Pro Leu Trp Asp Arg LeuAsp Tyr Asn Trp Phe Gln Lys Leu Pro 35 40 45 Asp Phe Phe Ser Lys Arg ValGlu Lys Tyr Asn Ser Thr Val Phe Arg 50 55 60 Thr Asn Val Pro Pro Cys PhePro Phe Phe Leu Gly Val Asn Pro Asn 65 70 75 80 Val Val Ala Val Leu AspVal Lys Ser Phe Ala His Leu Phe Asp Met 85 90 95 Glu Ile Val Glu Lys AlaAsn Val Leu Val Gly Asp Phe Met Pro Ser 100 105 110 Val Val Tyr Thr GlyAsp Met Arg Val Cys Ala Tyr Leu Asp Thr Ser 115 120 125 Glu Pro Lys HisThr Gln Ile Lys Asn Phe Ser Leu Asp Ile Leu Lys 130 135 140 Arg Ser SerLys Thr Trp Val Pro Thr Leu Val Lys Glu Leu Asp Thr 145 150 155 160 LeuPhe Gly Thr Phe Glu Ser Asp Leu Ser Lys Ser Lys Ser Ala Ser 165 170 175Leu Leu Pro Ala Leu Gln Lys Phe Leu Phe Asn Phe Phe Ser Leu Thr 180 185190 Phe Leu Gly Ala Asp Pro Ser Ala Ser Pro Glu Ile Ala Asn Ser Gly 195200 205 Phe Ala Tyr Leu Asp Ala Trp Leu Ala Ile Gln Leu Ala Pro Thr Val210 215 220 Ser Ile Gly Val Leu Gln Pro Leu Glu Glu Ile Phe Val His SerPhe 225 230 235 240 Ser Tyr Pro Tyr Phe Leu Val Arg Gly Gly Tyr Glu LysLeu Ile Lys 245 250 255 Phe Val Lys Ser Glu Ala Lys Glu Val Leu Thr ArgAla Gln Thr Asp 260 265 270 Phe Gln Leu Thr Glu Gln Glu Ala Ile His AsnLeu Leu Phe Ile Leu 275 280 285 Gly Phe Asn Ala Phe Gly Gly Phe Thr IlePhe Leu Pro Thr Leu Leu 290 295 300 Gly Asn Leu Gly Asp Glu Lys Asn AlaGlu Met Gln Glu Lys Leu Arg 305 310 315 320 Lys Glu Val Arg Glu Lys ValGly Thr Asn Gln Glu Asn Leu Ser Phe 325 330 335 Glu Ser Val Lys Glu MetGlu Leu Val Gln Ser Phe Val Tyr Glu Ser 340 345 350 Leu Arg Leu Ser ProPro Val Pro Ser Gln Tyr Ala Arg Ala Arg Lys 355 360 365 Asp Phe Met LeuSer Ser His Asp Ser Val Tyr Glu Ile Lys Lys Gly 370 375 380 Glu Leu LeuCys Gly Tyr Gln Pro Leu Val Met Lys Asp Pro Lys Val 385 390 395 400 PheAsp Glu Pro Glu Lys Phe Met Leu Glu Arg Phe Thr Lys Glu Lys 405 410 415Gly Lys Glu Leu Leu Asn Tyr Leu Phe Trp Ser Asn Gly Pro Gln Thr 420 425430 Gly Ser Pro Thr Glu Ser Asn Lys Gln Cys Ala Ala Lys Asp Ala Val 435440 445 Thr Leu Thr Ala Ser Leu Ile Val Ala Tyr Ile Phe Gln Lys Tyr Asp450 455 460 Ser Val Ser Phe Ser Ser Gly Ser Leu Thr Ser Val Lys Lys AlaCys 465 470 475 480 <210> SEQ ID NO 33 <211> LENGTH: 483 <212> TYPE: PRT<213> ORGANISM: Banana <400> SEQUENCE: 33 Met Ala Met Met Trp Ser SerAla Ser Ala Thr Ala Val Thr Thr Leu 1 5 10 15 Pro Thr Arg Pro Ile ProGly Ser Tyr Gly Pro Pro Leu Val Gly Pro 20 25 30 Leu Lys Asp Arg Leu AspTyr Phe Trp Phe Gln Gly Pro Glu Thr Phe 35 40 45 Phe Arg Ser Arg Met AlaThr His Lys Ser Thr Val Phe Arg Thr Asn 50 55 60 Met Pro Pro Thr Phe ProPhe Phe Val Gly Val Asp Pro Arg Val Val 65 70 75 80 Thr Val Leu Asp CysThr Ser Phe Ser Ala Leu Phe Asp Leu Glu Val 85 90 95 Val Glu Lys Lys AsnIle Leu Ile Gly Asp Tyr Met Pro Ser Leu Ser 100 105 110 Phe Thr Gly AspThr Arg Val Val Val Tyr Leu Asp Pro Ser Glu Pro 115 120 125 Asp His AlaArg Val Lys Ser Phe Cys Leu Glu Leu Leu Arg Arg Gly 130 135 140 Ala LysThr Trp Val Ser Ser Phe Leu Ser Asn Leu Asp Val Met Leu 145 150 155 160Ala Thr Ile Glu Gln Gly Ile Ala Lys Asp Gly Ser Ala Gly Leu Phe 165 170175 Gly Pro Leu Gln Lys Cys Ile Phe Ala Phe Leu Cys Lys Ser Ile Ile 180185 190 Gly Ala Asp Pro Ser Val Ser Pro Asp Val Gly Glu Asn Gly Phe Val195 200 205 Met Leu Asp Lys Trp Leu Ala Leu Gln Leu Leu Pro Thr Val LysVal 210 215 220 Gly Ala Ile Pro Gln Pro Leu Glu Glu Ile Leu Leu His SerPhe Pro 225 230 235 240 Leu Pro Phe Phe Leu Val Ser Arg Asp Tyr Arg LysLeu Tyr Glu Phe 245 250 255 Val Glu Lys Gln Gly Gln Glu Val Val Arg ArgAla Glu Thr Glu His 260 265 270 Gly Leu Ser Lys His Asp Ala Ile Asn AsnIle Leu Phe Val Leu Gly 275 280 285 Phe Asn Ala Phe Gly Gly Phe Ser ValPhe Phe Pro Thr Leu Leu Thr 290 295 300 Thr Ile Gly Arg Asp Lys Thr GlyLeu Arg Glu Lys Leu Lys Asp Glu 305 310 315 320 Val Arg Arg Val Met LysSer Arg Gly Glu Lys Arg Pro Ser Phe Glu 325 330 335 Thr Val Arg Glu MetGlu Leu Val Arg Ser Thr Val Tyr Glu Val Leu 340 345 350 Arg Leu Asn ProPro Val Pro Leu Gln Tyr Gly Arg Ala Arg Thr Asp 355 360 365 Phe Thr LeuAsn Ser His Asp Ala Ala Phe Lys Val Glu Lys Gly Glu 370 375 380 Leu LeuCys Gly Tyr Gln Pro Leu Val Met Arg Asp Pro Ala Val Phe 385 390 395 400Asp Asp Pro Glu Thr Phe Ala Pro Glu Arg Phe Met Gly Ser Gly Lys 405 410415 Glu Leu Leu Lys Tyr Val Phe Trp Ser Asn Gly Pro Glu Thr Gly Thr 420425 430 Pro Thr Pro Ala Asn Lys Gln Cys Ala Ala Lys Asp Tyr Val Val Glu435 440 445 Thr Ala Cys Leu Leu Met Ala Glu Ile Phe Tyr Arg Tyr Asp GluPhe 450 455 460 Val Cys Ala Asp Asp Ala Ile Ser Val Thr Lys Leu Asp ArgAla Arg 465 470 475 480 Glu Trp Glu <210> SEQ ID NO 34 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence:/Note = syntheticconstruct <400> SEQUENCE: 34 ggttatcagc cgctggtgat g 21 <210> SEQ ID NO35 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence:/Note = synthetic construct <400> SEQUENCE: 35 atgaaccggaggcgtttaat ccg 23 <210> SEQ ID NO 36 <211> LENGTH: 23 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence:/Note = syntheticconstruct <400> SEQUENCE: 36 acagagcgga cgagttcgta cct 23 <210> SEQ IDNO 37 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence:/Note = synthetic construct <400> SEQUENCE: 37aggattcgga gaagttcgtg ggc 23 <210> SEQ ID NO 38 <211> LENGTH: 488 <212>TYPE: PRT <213> ORGANISM: Psidium guava <400> SEQUENCE: 38 Met Ala ArgVal Val Met Ser Asn Met Ser Pro Ala Met Ser Ser Thr 1 5 10 15 Tyr ProPro Ser Leu Ser Pro Pro Ser Ser Pro Arg Pro Thr Thr Leu 20 25 30 Pro ValArg Thr Ile Pro Gly Ser Tyr Gly Trp Pro Leu Leu Gly Pro 35 40 45 Ile SerAsp Arg Leu Asp Tyr Phe Trp Phe Gln Gly Pro Glu Thr Phe 50 55 60 Phe ArgLys Arg Ile Glu Lys Tyr Lys Ser Thr Val Phe Arg Ala Asn 65 70 75 80 ValPro Pro Cys Phe Pro Phe Phe Ser Asn Val Asn Pro Asn Val Val 85 90 95 ValVal Leu Asp Cys Glu Ser Phe Ala His Leu Phe Asp Met Glu Ile 100 105 110Val Glu Lys Ser Asn Val Leu Val Gly Asp Phe Met Pro Ser Val Lys 115 120125 Tyr Thr Gly Asn Ile Arg Val Cys Ala Tyr Leu Asp Thr Ser Glu Pro 130135 140 Gln His Ala Gln Val Lys Asn Phe Ala Met Asp Ile Leu Lys Arg Ser145 150 155 160 Ser Lys Val Trp Glu Ser Glu Val Ile Ser Asn Leu Asp ThrMet Trp 165 170 175 Asp Thr Ile Glu Ser Ser Leu Ala Lys Asp Gly Asn AlaSer Val Ile 180 185 190 Phe Pro Leu Gln Lys Phe Leu Phe Asn Phe Leu SerLys Ser Ile Ile 195 200 205 Gly Ala Asp Pro Ala Ala Ser Pro Gln Val AlaLys Ser Gly Tyr Ala 210 215 220 Met Leu Asp Arg Trp Leu Ala Leu Gln LeuLeu Pro Thr Ile Asn Ile 225 230 235 240 Gly Val Leu Gln Pro Leu Val GluIle Phe Leu His Ser Trp Ala Tyr 245 250 255 Pro Phe Ala Leu Val Ser GlyAsp Tyr Asn Lys Leu Tyr Gln Phe Ile 260 265 270 Glu Lys Glu Gly Arg GluAla Val Glu Arg Ala Lys Ala Glu Phe Gly 275 280 285 Leu Thr His Gln GluAla Ile His Asn Leu Leu Phe Ile Leu Gly Phe 290 295 300 Asn Ala Phe GlyGly Phe Ser Ile Phe Leu Pro Thr Leu Leu Ser Asn 305 310 315 320 Ile LeuSer Asp Thr Thr Gly Leu Gln Asp Arg Leu Arg Lys Glu Val 325 330 335 ArgAla Lys Gly Gly Pro Ala Leu Ser Phe Ala Ser Val Lys Glu Met 340 345 350Glu Leu Val Lys Ser Val Val Tyr Glu Thr Leu Arg Leu Asn Pro Pro 355 360365 Val Pro Phe Gln Tyr Ala Arg Ala Arg Lys Asp Phe Gln Leu Lys Ser 370375 380 His Asp Ser Val Phe Asp Val Lys Lys Gly Glu Leu Leu Cys Gly Tyr385 390 395 400 Gln Lys Val Val Met Thr Asp Pro Lys Val Phe Asp Glu ProGlu Ser 405 410 415 Phe Asn Ser Asp Arg Phe Val Gln Asn Ser Glu Leu LeuAsp Tyr Leu 420 425 430 Tyr Trp Ser Asn Gly Pro Gln Thr Gly Thr Pro ThrGlu Ser Asn Lys 435 440 445 Gln Cys Ala Ala Lys Asp Tyr Val Thr Leu ThrAla Cys Leu Phe Val 450 455 460 Ala Tyr Met Phe Arg Arg Tyr Asn Ser ValThr Gly Ser Ser Ser Ser 465 470 475 480 Ile Thr Ala Val Glu Lys Ala Asn485 <210> SEQ ID NO 39 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence:/Note = synthetic construct <400> SEQUENCE: 39ccgtcagcac caccaaatcc ttc 23 <210> SEQ ID NO 40 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence:/Note = syntheticconstruct <400> SEQUENCE: 40 gaacagataa tccagcaggg c 21 <210> SEQ ID NO41 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence:/Note = synthetic construct <400> SEQUENCE: 41 ctgaaccgaccgcgactgtg t 21 <210> SEQ ID NO 42 <211> LENGTH: 22 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence:/Note = syntheticconstruct <400> SEQUENCE: 42 tcgcccgtga accgatcagg ta 22 <210> SEQ ID NO43 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence:/Note = synthetic construct <400> SEQUENCE: 43 tccgcgtcggctccactgtc 20 <210> SEQ ID NO 44 <211> LENGTH: 22 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence:/Note = synthetic construct <400>SEQUENCE: 44 tctcccacga acctatcgcc ca 22 <210> SEQ ID NO 45 <211>LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence:/Note = synthetic construct <400> SEQUENCE: 45 gccatggcctccattgtcat tccttc 26 <210> SEQ ID NO 46 <211> LENGTH: 45 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence:/Note = syntheticconstruct <400> SEQUENCE: 46 ggaattctta gtgatggtga tggtgatgga aacttgctttcttag 45 <210> SEQ ID NO 47 <211> LENGTH: 27 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence:/Note = synthetic construct <400>SEQUENCE: 47 gcatatggct actccttctt cctcctc 27 <210> SEQ ID NO 48 <211>LENGTH: 46 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence:/Note = synthetic construct <400> SEQUENCE: 48 catcgatttagtgatggtga tggtgatgat tagtcattag ctttaa 46 <210> SEQ ID NO 49 <211>LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence:/Note = synthetic construct <400> SEQUENCE: 49 atgaattcggtacccgggat cctttttttt ttttttttt 39 <210> SEQ ID NO 50 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence:/Note = syntheticconstruct <400> SEQUENCE: 50 atgaattcgg tacccgggat c 21 <210> SEQ ID NO51 <211> LENGTH: 1596 <212> TYPE: DNA <213> ORGANISM: Cucumis melo <400>SEQUENCE: 51 atgtcctcca ttgtcattcc ttctcttcaa cctcacttgc gattcccatcctcgcaagaa 60 acgcctcaaa gatctcgttc tagagttggc ttcgtttcca tacgtccaatctacgccacc 120 gacggagttt cttcctcgtc ttcttcctct cttcaagtgc cgcagcggattgtttcgccg 180 ccggaaccca ccaagcttcc tttgaggaag gttcccggtg attatgggccaccgatgttt 240 ggggcgttga aggacagaca tgattatttt tataatcagg ggagggaagagtatttgaaa 300 tctcgaatgc tccggtatga atccactgtg tatagaacta atatgccgccgggtccattt 360 atcacttccg attcccgagt tgttgtttta ctcgacggga agagttttcctgttcttttc 420 gaccattcta aagttgagaa gaaagatctc tttatcggaa cttacatgcctgtaacagag 480 ctcaccggcg gttacagggt gctttcttat attgacccat ctgagcccgatcacgctaag 540 cttaaacagt tgattttctt tctcctcaag caccgccggg ataaaattatgccggaattt 600 cactctactt tttctgagct attcgagact ctggaaaagg atttggctgctgctggtaga 660 gcagagtaca atgcttccgg tgaacaagcg gcgtttaatt tcttggctcggtctcttttc 720 ggcgctgatc cggtagattc caaattgggt cgcgatgcgc cgaaattgatcgcgaaatgg 780 gtcttattcc agcttggccc tgttctgagt ctcggcctcc ccaaggtcgtcgaggagctt 840 ctcctccgca cggtccggct ccccccggcg ttgattaaag ccgattaccgtcggttgtac 900 gacttctttt acaagtcgtc ggaggcggtg tttgaggagg cggatagattgggaatttcg 960 agggaagaag cttgtcacaa cttgctattc acaacttgtt ttaattcatttggagggatg 1020 aagatctttt tccccaatat gataaaatgg atcggccgag ccggagtgaatctccacacc 1080 cgactagcac gggagattcg tactgccgta aaagccaacg gcgggaaaatcacgatgggg 1140 gctatggaac agatgccgct gatgaaatca gtggtgtacg aagcgttaagaatcgagccg 1200 ccggttccgg ttcagtacgg tcgggcaaag aaagaccttg tggtggaaagccacgacgcg 1260 gctttcgaga tcaaagaagg agaagtgatt tgtgggtatc agccattcgcaacaagagat 1320 ccgaaaatct tcgacagagc ggacgagttc gtacctgatc ggttcacgggcgagggtgag 1380 gagcttctca aacacgtcat atggtcaaac ggaccggaaa cacagtcgccgtcggttcag 1440 aacaagcagt gcgcaggaaa agacttcatc gtcttcatct ctcggcttctcgtcgttgaa 1500 cttttcctcc gttacgactc cttcgacatc gaagcctcaa acactccgttaggtgccgcc 1560 gtcaccgtaa cctccctaaa gaaagcaagt ttctaa 1596 <210> SEQID NO 52 <211> LENGTH: 465 <212> TYPE: PRT <213> ORGANISM: Cucumis melo<400> SEQUENCE: 52 Asn Asp Met Ser Ser Ile Val Ile Pro Ser Leu Gln ProHis Leu Arg 1 5 10 15 Phe Pro Ser Ser Gln Glu Thr Pro Gln Arg Ser ArgSer Arg Val Gly 20 25 30 Phe Val Ser Ile Arg Pro Ile Tyr Ala Thr Asp GlyVal Ser Ser Ser 35 40 45 Ser Ser Ser Ser Leu Gln Val Pro Gln Arg Ile ValSer Pro Pro Glu 50 55 60 Pro Thr Lys Leu Pro Leu Arg Lys Val Pro Gly AspTyr Gly Pro Pro 65 70 75 80 Met Phe Gly Ala Leu Lys Asp Arg His Asp TyrPhe Tyr Asn Gln Gly 85 90 95 Arg Glu Glu Tyr Leu Lys Ser Arg Met Leu ArgTyr Glu Ser Thr Val 100 105 110 Tyr Arg Thr Asn Met Pro Pro Gly Pro PheIle Thr Ser Asp Ser Arg 115 120 125 Val Val Val Leu Leu Asp Gly Lys SerPhe Pro Val Leu Phe Asp His 130 135 140 Ser Lys Val Glu Lys Lys Asp LeuPhe Thr Gly Ala Val Phe Glu Glu 145 150 155 160 Ala Asp Arg Leu Gly IleSer Arg Glu Glu Ala Cys His Asn Leu Leu 165 170 175 Phe Thr Thr Cys PheAsn Ser Phe Gly Gly Met Lys Ile Phe Phe Pro 180 185 190 Asn Met Ile LysTrp Ile Gly Arg Ala Gly Val Asn Leu His Thr Arg 195 200 205 Leu Ala ArgGlu Ile Arg Thr Ala Val Lys Ala Asn Gly Gly Lys Ile 210 215 220 Thr MetGly Ala Met Glu Gln Met Pro Leu Met Lys Ser Val Val Tyr 225 230 235 240Glu Ala Leu Arg Ile Glu Pro Pro Val Pro Val Gln Tyr Gly Arg Ala 245 250255 Lys Lys Asp Leu Val Val Glu Ser His Asp Ala Ala Phe Glu Ile Lys 260265 270 Glu Gly Glu Val Ile Cys Gly Tyr Gln Pro Phe Ala Thr Arg Asp Pro275 280 285 Lys Ile Phe Asp Arg Ala Asp Glu Leu Val Pro Asp Arg Phe ThrGly 290 295 300 Glu Gly Glu Glu Leu Leu Thr Tyr Met Pro Val Thr Glu LeuThr Gly 305 310 315 320 Gly Tyr Arg Val Leu Ser Tyr Ile Asp Pro Ser GluPro Asp His Ala 325 330 335 Lys Leu Lys Gln Leu Ile Phe Phe Leu Leu LysHis Arg Arg Asp Lys 340 345 350 Ile Met Pro Glu Phe His Ser Thr Phe SerGlu Leu Phe Glu Thr Leu 355 360 365 Glu Lys Asp Leu Ala Ala Ala Gly ArgAla Glu Tyr Asn Ala Ser Gly 370 375 380 Glu Gln Ala Ala Phe Asn Phe LeuAla Arg Ser Leu Phe Gly Ala Asp 385 390 395 400 Pro Val Asp Ser Lys LeuGly Arg Asp Ala Pro Lys Leu Ile Ala Lys 405 410 415 Trp Val Leu Phe GlnLeu Gly Pro Val Leu Ser Leu Gly Leu Pro Lys 420 425 430 Val Val Glu GluLeu Leu Leu Arg Thr Val Arg Leu Pro Pro Ala Leu 435 440 445 Ile Lys AlaAsp Tyr Arg Arg Leu Tyr Asp Phe Phe Tyr Lys Ser Ser 450 455 460 Glu 465<210> SEQ ID NO 53 <211> LENGTH: 468 <212> TYPE: PRT <213> ORGANISM:Flax <400> SEQUENCE: 53 Met Ala Ser Ser Ala Leu Asn Asn Leu Val Ala ValAsn Pro Asn Thr 1 5 10 15 Leu Ser Pro Ser Pro Lys Ser Thr Pro Leu ProAsn Thr Phe Ser Asn 20 25 30 Leu Arg Arg Val Ser Ala Phe Arg Pro Ile LysAla Ser Leu Phe Gly 35 40 45 Asp Ser Pro Ile Lys Ile Pro Gly Ile Thr SerGln Pro Pro Pro Ser 50 55 60 Ser Asp Glu Thr Thr Leu Pro Ile Arg Gln IlePro Gly Asp Tyr Gly 65 70 75 80 Leu Pro Gly Ile Gly Pro Ile Gln Asp ArgLeu Asp Tyr Phe Tyr Asn 85 90 95 Gln Gly Arg Glu Glu Phe Phe Lys Ser ArgLeu Gln Lys Tyr Lys Ser 100 105 110 Thr Val Tyr Arg Ala Asn Met Pro ProGly Pro Phe Ile Ala Ser Asn 115 120 125 Pro Arg Val Ile Val Leu Leu AspAla Lys Ser Phe Pro Val Leu Phe 130 135 140 Asp Met Ser Lys Val Glu LysLys Asp Leu Phe Thr Gly Ser Val Leu 145 150 155 160 Asp Glu Ala Glu GlnSer Gly Ile Ser Arg Asp Glu Ala Cys His Asn 165 170 175 Ile Leu Phe AlaVal Cys Phe Asn Ser Trp Gly Gly Phe Lys Ile Leu 180 185 190 Phe Pro SerLeu Met Lys Trp Ile Gly Arg Ala Gly Leu Glu Leu His 195 200 205 Thr LysLeu Ala Gln Glu Ile Arg Ser Ala Ile Gln Ser Thr Gly Gly 210 215 220 GlyLys Val Thr Met Ala Ala Met Glu Gln Met Pro Leu Met Lys Ser 225 230 235240 Val Val Tyr Glu Thr Leu Arg Ile Glu Pro Pro Val Ala Leu Gln Tyr 245250 255 Gly Lys Ala Lys Lys Asp Phe Ile Leu Glu Ser His Glu Ala Ala Tyr260 265 270 Gln Val Lys Glu Gly Glu Met Leu Phe Gly Tyr Gln Pro Phe AlaThr 275 280 285 Lys Asp Pro Lys Ile Phe Asp Arg Pro Glu Glu Phe Val AlaAsp Arg 290 295 300 Phe Val Gly Glu Gly Val Lys Leu Met Thr Tyr Met ProSer Thr Glu 305 310 315 320 Leu Thr Gly Gly Tyr Arg Ile Leu Ser Tyr LeuAsp Pro Ser Glu Pro 325 330 335 Asn His Thr Lys Leu Lys Gln Leu Leu PheAsn Leu Ile Lys Asn Arg 340 345 350 Arg Asp Tyr Val Ile Pro Glu Phe SerSer Ser Phe Thr Asp Leu Cys 355 360 365 Glu Val Val Glu Tyr Asp Leu AlaThr Lys Gly Lys Ala Ala Phe Asn 370 375 380 Asp Pro Ala Glu Gln Ala AlaPhe Asn Phe Leu Ser Arg Ala Phe Phe 385 390 395 400 Gly Val Lys Pro IleAsp Thr Pro Leu Gly Lys Asp Ala Pro Ser Leu 405 410 415 Ile Ser Lys TrpVal Leu Phe Asn Leu Ala Pro Ile Leu Ser Val Gly 420 425 430 Leu Pro LysGlu Val Glu Glu Ala Thr Leu His Ser Val Arg Leu Pro 435 440 445 Pro LeuLeu Val Gln Asn Asp Tyr His Arg Leu Tyr Glu Phe Phe Thr 450 455 460 SerAla Ala Gly 465 <210> SEQ ID NO 54 <211> LENGTH: 405 <212> TYPE: PRT<213> ORGANISM: Guayule <400> SEQUENCE: 54 Met Asp Pro Ser Ser Lys ProLeu Arg Glu Ile Pro Gly Ser Tyr Gly 1 5 10 15 Ile Pro Phe Phe Gln ProIle Lys Asp Arg Leu Glu Tyr Phe Tyr Gly 20 25 30 Thr Gly Gly Arg Asp GluTyr Phe Arg Ser Arg Met Gln Lys Tyr Gln 35 40 45 Ser Thr Val Phe Arg AlaAsn Met Pro Pro Gly Pro Phe Val Ser Ser 50 55 60 Asn Pro Lys Val Ile ValLeu Leu Asp Ala Lys Ser Phe Pro Ile Leu 65 70 75 80 Phe Asp Val Ser LysVal Glu Lys Lys Asp Leu Phe Thr Gly Pro Val 85 90 95 Met Glu Gln Ala GluLys Leu Gly Val Pro Lys Asp Glu Ala Val His 100 105 110 Asn Ile Leu PheAla Val Cys Phe Asn Thr Phe Gly Gly Val Lys Ile 115 120 125 Leu Phe ProAsn Thr Leu Lys Trp Ile Gly Val Ala Gly Glu Asn Leu 130 135 140 His ThrGln Leu Ala Glu Glu Ile Arg Gly Ala Ile Lys Ser Tyr Gly 145 150 155 160Asp Gly Asn Val Thr Leu Glu Ala Ile Glu Gln Met Pro Leu Thr Lys 165 170175 Ser Val Val Tyr Glu Ser Leu Arg Ile Glu Pro Pro Val Pro Pro Gln 180185 190 Tyr Gly Lys Ala Lys Ser Asn Phe Thr Ile Glu Ser His Asp Ala Thr195 200 205 Phe Glu Val Lys Lys Gly Glu Met Leu Phe Gly Tyr Gln Pro PheAla 210 215 220 Thr Lys Asp Pro Lys Val Phe Asp Arg Pro Glu Glu Phe ValPro Asp 225 230 235 240 Arg Phe Val Gly Asp Gly Glu Ala Leu Leu Thr TyrMet Pro Ser Thr 245 250 255 Lys Leu Thr Gly Ala Tyr Arg Val Leu Ser TyrLeu Asp Pro Ser Glu 260 265 270 Pro Arg His Ala Gln Leu Lys Asn Leu LeuPhe Phe Met Leu Lys Asn 275 280 285 Ser Ser Asn Arg Val Ile Pro Gln PheGlu Thr Thr Tyr Thr Glu Leu 290 295 300 Phe Glu Gly Leu Glu Ala Glu LeuAla Lys Asn Gly Lys Ala Ala Phe 305 310 315 320 Asn Asp Val Gly Glu GlnAla Ala Phe Arg Phe Leu Gly Arg Ala Tyr 325 330 335 Phe Asn Ser Asn ProGlu Glu Thr Lys Leu Gly Thr Ser Ala Pro Thr 340 345 350 Leu Ile Ser SerTrp Val Leu Phe Asn Leu Ala Pro Thr Leu Asp Leu 355 360 365 Gly Leu ProTrp Phe Leu Gln Glu Pro Leu Leu His Thr Phe Arg Leu 370 375 380 Pro AlaPhe Leu Ile Lys Ser Thr Tyr Asn Lys Leu Tyr Asp Tyr Phe 385 390 395 400Gln Ser Val Ala Thr 405 <210> SEQ ID NO 55 <211> LENGTH: 448 <212> TYPE:PRT <213> ORGANISM: Arabidopsis <400> SEQUENCE: 55 Met Ala Ser Ile SerThr Pro Phe Pro Ile Ser Leu His Pro Lys Thr 1 5 10 15 Val Arg Ser LysPro Leu Lys Phe Arg Val Leu Thr Arg Pro Ile Lys 20 25 30 Ala Ser Gly SerGlu Thr Pro Asp Leu Thr Val Ala Thr Arg Thr Gly 35 40 45 Ser Lys Asp LeuPro Ile Arg Asn Ile Pro Gly Asn Tyr Gly Leu Pro 50 55 60 Ile Val Gly ProIle Lys Asp Arg Trp Asp Tyr Phe Tyr Asp Gln Gly 65 70 75 80 Ala Glu GluPhe Phe Lys Ser Arg Ile Arg Lys Tyr Asn Ser Thr Val 85 90 95 Tyr Arg ValAsn Met Pro Pro Gly Ala Phe Ile Ala Glu Asn Pro Gln 100 105 110 Val ValAla Leu Leu Asp Gly Lys Ser Phe Pro Val Leu Phe Asp Val 115 120 125 AspLys Val Glu Lys Lys Asp Leu Phe Thr Gly Glu Ile Leu Val Glu 130 135 140Ala Asp Lys Leu Gly Ile Ser Arg Glu Glu Ala Thr His Asn Leu Leu 145 150155 160 Phe Ala Thr Ser Phe Asn Thr Trp Gly Gly Met Lys Ile Leu Phe Pro165 170 175 Asn Met Val Lys Arg Ile Gly Pro Gly Gly His Gln Val His AsnArg 180 185 190 Leu Ala Glu Glu Ile Arg Ser Val Ile Lys Ser Asn Gly GlyGlu Leu 195 200 205 Thr Met Gly Ala Ile Glu Lys Met Glu Leu Thr Lys SerVal Val Tyr 210 215 220 Glu Cys Leu Arg Phe Glu Pro Pro Val Thr Ala GlnTyr Gly Arg Ala 225 230 235 240 Lys Lys Asp Leu Val Ile Glu Ser His AspAla Ala Phe Lys Val Lys 245 250 255 Ala Gly Glu Met Leu Tyr Gly Tyr GlnPro Leu Ala Thr Arg Asp Pro 260 265 270 Lys Ile Phe Asp Arg Ala Asp GluPhe Val Pro Glu Arg Phe Val Gly 275 280 285 Glu Glu Gly Glu Lys Leu LeuThr Tyr Met Pro Ser Thr Glu Leu Thr 290 295 300 Gly Gly Tyr Arg Ile LeuSer Tyr Leu Asp Pro Ser Glu Pro Lys His 305 310 315 320 Glu Lys Leu LysAsn Leu Leu Phe Phe Leu Leu Lys Ser Ser Asn Arg 325 330 335 Ile Phe ProGlu Phe Gln Ala Thr Tyr Ser Glu Leu Phe Asp Ser Leu 340 345 350 Glu LysGlu Ala Phe Pro Leu Arg Glu Ser Gly Phe Arg Arg Phe Gln 355 360 365 ArgArg Asn Arg Leu Leu Phe Leu Gly Ser Ser Phe Leu Arg Asp Glu 370 375 380Ser Arg Arg Tyr Lys Leu Lys Ala Asp Ala Pro Gly Leu Ile Thr Lys 385 390395 400 Trp Val Leu Phe Asn Leu His Pro Leu Leu Ser Ile Gly Leu Pro Arg405 410 415 Val Ile Glu Glu Pro Leu Ile His Thr Phe Ser Leu Pro Pro AlaLeu 420 425 430 Val Lys Ser Asp Tyr Gln Arg Leu Tyr Glu Phe Leu Arg IleArg Gly 435 440 445 <210> SEQ ID NO 56 <211> LENGTH: 1715 <212> TYPE:DNA <213> ORGANISM: Cucumis melo <221> NAME/KEY: misc_feature <222>LOCATION: 1283 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE:56 atggctactc cttcttcctc ctcccctgaa cttcctctca aaccaattcc cggtggctat 60ggcttcccct tcctcggtcc catcaaagac cgttacgatt acttctattt ccaaggtaga 120gacgaattct tccgttcccg gattaccaaa tacaactcca ccgtcttccg cgccaacatg 180ccaccgggcc ccttcatttc ctccgattcc agagtcgttg tccttctcga tgccctcagt 240tttcctatcc tcttcgacac agccaaagtc gagaaacgca acattctcga cggaacttac 300atgccctcct tgtccttcac cggcaacatt cgcacctgtg cttatttgga cccatcggaa 360acagagcact ctgttctcaa acgcctcttc ctctcctttc tcgcttcccg ccatgacagg 420ttcatccctc tgtttcgaag ctccttgtct gagatgtttg ttaagcttga agataaactt 480tccgagaaaa agaagatcgc tgatttcaac tcgatcagcg attccatgtc gtttgattat 540gttttccgtt tactctccga tggaacccct gattcgaaat tagctgctga gggacctgga 600atgttcgatc tgtggcttgt gtttcaactc gccccattgg cttccattgg ccttcccaaa 660attttctctg tttttgaaga tctcgtcatt cacaccattc ccctgccttt cttcccagtc 720aagagtggtt acaggaagct ttatgaagcg ttttactcct cttctggctc atttctagac 780gaagcagaga aacaggggat agacagggag aaagcatgtc acaatttagt gtttctcgct 840ggattcaacg catacggggg aatgaaagtc ctttttccca ctttactgaa atgggtcggc 900accgccggcg aggatctcca ccggaaactc gccgaggaag tcaggacaac cgtgaaggaa 960gaagggggac tgactttctc cgccttggag aaaatgagtc tgctgaagtc cgtcgtgtac 1020gaagcactca ggatcgaacc gccggtgccg ttccagtacg ggaaagcgaa ggaggatatc 1080gtgattcaga gccacgattc ttctttcaag atcaaaaaag gggagacgat ttttggttat 1140cagccgtttg ctactaaaga tccgaagatt tttaaggatt cggagaagtt cgtgggcgat 1200aggttcgtgg gagaagaagg ggagaagctt ttgaagtatg tttactggtc aaatgagcgg 1260gagacagtgg agccgacgcg gangaacaag cagtgtccgg ggaagaatct ggtggtgctg 1320ataggtagga ttatggtggt ggaattcttc cttcgttatg atacgttcac cgtggaggtc 1380gcggatttgc cgctgggtcc ggcagtgaag ttcaagtcct taaccagagc aaccgatatg 1440ttaaagctaa tgactaatta gttttatcat ttacagatag tgaattggtt gatgcacgga 1500agctgtggcg gactgcgcac acatgattga gtacttgggg ttattaaagt aatttcgttg 1560tgatccacgt ggtcttattt taatttgaga tctcattgtg tgttgtaacc caccggtcat 1620cttattttat agtttgtttg ttttctcaat tatgctccaa attttaaaat aaataaatac 1680catcttcttc ttttactaaa aaaaaaaaaa aaaaa 1715

What is claimed is:
 1. An isolated nucleic acid that encodes a fattyacid hydroperoxide lyase present in melon, wherein the lyase hasactivity for both 9-hydroperoxide substrates and 13-hydroperoxidesubstrates and wherein K_(m) and V_(max) of the lyase for9-hydroperoxylinolenic acid are greater than K_(m) and V_(max) of thelyase for 9-hydroperoxylinoleic acid.
 2. The nucleic acid of claim 1,comprising the nucleic acid sequence set forth in SEQ ID NO:8.
 3. Anisolated nucleic acid claim 1 that encodes a lyase comprising an aminoacid sequence selected from the group consisting of SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.
 4. Avector, comprising the nucleic acid of claim
 1. 5. The vector of claim4, further comprising a promoter functionally linked to the nucleicacid.
 6. The vector of claim 4, wherein the vector is a plasmid.
 7. Acell containing an exogenous nucleic acid comprising the nucleic acid ofclaim
 1. 8. The cell of claim 7, wherein the cell is a prokaryotic cell.9. The cell of claim 8, wherein the prokaryotic cell is selected fromthe group consisting of an Escherichia coli cell, a Bacillus cell, and aStreptomyces cell.
 10. The cell of claim 7, wherein the cell is aeukaryotic cell.
 11. The cell of claim 10, wherein the eukaryotic cellis selected from the group consisting of a yeast cell, a plant cell, andan insect cell.